Therapies for hematologic malignancies

ABSTRACT

The invention provides methods that relate to a novel therapeutic strategy for the treatment of hematological malignancies and inflammatory diseases. In particular, the method comprises administering a compound of formula A, 
     
       
         
         
             
             
         
       
         
         
           
             wherein R is H, halo, or C1-C6 alkyl; 
             R′ is C1-C6 alkyl; or 
             a pharmaceutically acceptable salt thereof; and 
             optionally a pharmaceutically acceptable excipient; 
             and administering at least one additional therapeutic agent.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/277,857 filed Sep. 27, 2016, which is a divisional of U.S. patentapplication Ser. No. 13/417,185 filed Mar. 9, 2012, now U.S. Pat. No.9,492,449, issued on Nov. 15, 2016, which is a continuation-in-part ofU.S. patent application Ser. No. 12/618,612 filed Nov. 13, 2009, whichclaims priority from U.S. Provisional Patent Application Nos. 61/245,196filed Sep. 23, 2009; 61/231,278 filed Aug. 4, 2009; 61/180,768 filed May22, 2009; 61/155,057 filed Feb. 24, 2009; 61/142,845 filed Jan. 6, 2009;and 61/114,434 filed Nov. 13, 2008. The contents of these applicationsare incorporated by reference in their entirety.

TECHNICAL FIELD

The invention is in the field of therapeutics and medicinal chemistry.In particular, the invention concerns uses of certain quinazolinederivatives for the treatment of hematologic malignancies and certainother conditions.

BACKGROUND ART

Cell signaling via 3′-phosphorylated phosphoinositides has beenimplicated in a variety of cellular processes, e.g., malignanttransformation, growth factor signaling, inflammation, and immunity. Theenzyme responsible for generating these phosphorylated signalingproducts, phosphatidylinositol 3-kinase (PI 3-kinase; PI3K), wasoriginally identified as an activity associated with viral oncoproteinsand growth factor receptor tyrosine kinases that phosphorylatesphosphatidylinositol (PI) and its phosphorylated derivatives at the3′-hydroxyl of the inositol ring.

PI 3-kinase activation, is believed to be involved in a range ofcellular responses including cell growth, differentiation, andapoptosis.

The initial purification and molecular cloning of PI 3-kinase revealedthat it was a heterodimer consisting of p85 and p110 subunits. Fourdistinct Class I PI3Ks have been identified, designated PI3K α, β, δ,and γ, each consisting of a distinct 110 kDa catalytic subunit and aregulatory subunit. More specifically, three of the catalytic subunits,i.e., p110α, p110β and p110δ, each interact with the same regulatorysubunit, p85; whereas p110γ interacts with a distinct regulatorysubunit, p101. The patterns of expression of each of these PI3Ks inhuman cells and tissues are also distinct.

Identification of the p110δ isoform of PI 3-kinase is described inChantry et al., J Biol Chem, 272:19236-41 (1997). It was observed thatthe human p110δ isoform is expressed in a tissue-restricted fashion. Itis expressed at high levels in lymphocytes and lymphoid tissues,suggesting that the protein might play a role in PI 3-kinase-mediatedsignaling in the immune system. The p110β isoform of PI3K may also playa role in PI3K-mediated signaling in certain cancers.

There is a need for a treatment relating to PI3K mediated disordersrelating to cancers, inflammatory diseases, and autoimmune diseases.

SUMMARY

The present invention provides a class of quinazolinone type compoundsand a method to use these compounds in the treatment of cancer,inflammatory, and autoimmune diseases. In particular, cancers that arehematologic malignancies, such as leukemia and lymphoma, are treated bythe methods herein. Also provided are methods of using the quinazolinonecompounds in combination with other therapeutic treatments in patientsin need thereof.

In one aspect, the invention provides the use of a compound for themanufacture of a medicament for the treatment of a condition in asubject, wherein the condition is cancer or an autoimmune condition;wherein the compound is of formula A,

wherein R is H, halo, or C1-C6 alkyl; R′ is C1-C6 alkyl; or apharmaceutically acceptable salt thereof; and optionally apharmaceutically acceptable excipient.

In one embodiment, the compound is predominantly the S-enantiomer.

In some of the foregoing embodiments, R is fluoro (F) and is attached toposition 5 or 6 of the quinazolinyl ring.

In some of the foregoing embodiments, R is H or F; and R′ is methyl,ethyl or propyl.

In some embodiments, the compound is

In some embodiments, compound is

In some of the foregoing embodiments, the autoimmune disease is allergicrhinitis, asthma, COPD, or rheumatoid arthritis.

In some of the foregoing embodiments, the condition is cancer.

In some of the foregoing embodiments, the cancer is a hematologicalmalignancy.

In some of the foregoing embodiments, the hematological malignancy isleukemia.

In some of the foregoing embodiments, the hematological malignancy islymphoma.

In some of the foregoing embodiments, the hematological malignancy isselected from the group consisting of acute lymphocytic leukemia (ALL),acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), smalllymphocytic lymphoma (SLL), multiple myeloma (MM), non-Hodgkin'slymphoma (NHL), mantle cell lymphoma (MCL), follicular lymphoma,Waldenstrom's macroglobulinemia (WM), B-cell lymphoma and diffuse largeB-cell lymphoma (DLBCL).

In some of the foregoing embodiments, the cancer is acute lymphocyticleukemia (ALL).

In some of the foregoing embodiments, the cancer is acute myeloidleukemia (AML).

In some of the foregoing embodiments, the cancer is chronic lymphocyticleukemia (CLL).

In some of the foregoing embodiments, the cancer is multiple myeloma(MM).

In some of the foregoing embodiments, the cancer is B-cell lymphoma.

In some of the foregoing embodiments, the cancer is diffuse large B-celllymphoma (DLBCL).

In some of the foregoing embodiments, the cancer is B-cell or T-cellALL.

In some of the foregoing embodiments, the cancer is Hodgkin's lymphoma.

In some of the foregoing embodiments, the cancer is breast, lung, colon,prostate or ovarian cancer.

In some of the foregoing embodiments, the subject is refractory tochemotherapy treatment, or in relapse after treatment with chemotherapy.

In some of the foregoing embodiments, the compound is prepared foradministration with at least one additional therapeutic agent.

In some of the foregoing embodiments, the additional therapeutic agentis a proteasome inhibitor.

In some of the foregoing embodiments, the additional therapeutic agentis combined with the compound of Formula A.

In some of the foregoing embodiments, the additional therapeutic agentis selected from the group consisting of ofatumumab, bortezomib(Velcade®), carfilzomib (PR-171), PR-047, disulfiram, lactacystin,PS-519, eponemycin, epoxomycin, aclacinomycin, CEP-1612, MG-132, CVT63417, PS-341, vinyl sulfone tripeptide inhibitors, ritonavir, PI-083,(+/−) 7 methylomuralide, (−)-7-methylomuralide.

In some of the foregoing embodiments, the additional therapeutic agentis bortezomib.

In some of the foregoing embodiments, the compound is prepared foradministration with at least a group of at least two agents, whereinsaid group of agents is selected from the groups consisting of a-q,

a) CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone);

b) R CHOP (rituximab CHOP);

c) hyperCVAD (hyperfractionated cyclophosphamide, vincristine,doxorubicin, dexamethasone, methotrexate, cytarabine);

d) R-hyperCVAD (rituximab-hyperCVAD);

e) FCM (fludarabine, cyclophosphamide, mitoxantrone);

f) R-FCM (rituximab, fludarabine, cyclophosphamide, mitoxantrone);

g) bortezomib and rituximab;

h) temsirolimus and rituximab;

i) temsirolimus and Velcade®;

j) Iodine-131 tositumomab (Bexxar®) and CHOP;

k) CVP (cyclophosphamide, vincristine, prednisone);

l) R-CVP (rituximab-CVP);

m) ICE (iphosphamide, carboplatin, etoposide);

n) R-ICE (rituximab-ICE);

o) FCR (fludarabine, cyclophosphamide, rituximab);

p) FR (fludarabine, rituximab); and

q) D.T. PACE (dexamethasone, thalidomide, cisplatin, Adriamycin®,cyclophosphamide, etoposide).

In some of the foregoing embodiments, the compound of formula A ispresent in a pharmaceutical composition comprising the compound offormula A and at least one pharmaceutically acceptable excipient.

In another aspect, the invention provides the use of a compound for themanufacture of a medicament for the treatment of a condition in asubject, wherein the condition is selected from the group consisting ofmultiple myeloma, acute lymphocytic leukemia (ALL), acute myeloidleukemia (AML), chronic lymphocytic leukemia (CLL), B-cell lymphoma,diffuse large B-cell lymphoma (DLBCL), B cell ALL, T cell ALL, Hodgkin'slymphoma, breast, and ovarian cancer, wherein the compound is a compoundof formula I″ or formula II″:

In some of the foregoing embodiments, the subject is refractory tochemotherapy treatment or in relapse after treatment with chemotherapy.

In some of the foregoing embodiments, the subject has a cancer thatconstitutively expresses Akt phosphorylation activity.

In some of the foregoing embodiments, the subject has a cancer with highp110δ activity and low p110α activity.

In some of the foregoing embodiments, the compound is used incombination with bortezomib.

In another aspect, the invention provides the use of a compound I″ orII″ in the manufacture of a medicament for treating a hematologicalcancer, wherein the medicament is prepared for administration withbortezomib or carfilzomib.

In some of the foregoing embodiments, the compound maintains an averageblood concentration above the EC50 level for PI3Kδ activation and belowthe level for EC50 PI3Kγ activation in basophils over a period of atleast 12 hours from compound administration.

In some of the foregoing embodiments, the compound maintains an averageblood plasma concentration between 100 nM and 1100 nM over a period ofat least 12 hours from compound administration.

In some of the foregoing embodiments, the subject is resistant tostandard chemotherapeutic treatments.

In some of the foregoing embodiments, the subject has at least oneenlarged lymph node.

In some of the foregoing embodiments, the subject is refractory to atleast two standard or experimental chemotherapy treatments had at leasttwo prior chemotherapy treatments.

In some of the foregoing embodiments, each chemotherapy treatment isselected from the group consisting of fludarabine, alkylating agents,rituximab, alemtuzumab, and the treatments a-q listed above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a graphical summary of multiple myeloma (MM) cell growth asa function of varying concentrations of cytokines IGF-1 and IL-6 incombination with compound I, using LB cells.

FIG. 2 shows a graphical summary of cell growth of multiple myeloma (MM)cells as a function of varying concentrations of compound I and thepresence or absence of bone marrow stromal cells (BMSC) after 48 hours.

FIG. 3 shows a graphical summary of apoptosis of Chronic LymphocyticLeukemia (CLL) cells as a function of varying concentrations of compoundof formula I.

FIG. 4 shows a summary chart of the effect of compound I on cellviability, reduction in Akt (Ser473) phosphorylation, and caspase 3activation in several different Acute Lymphoblastic Leukemia (ALL) celllines.

FIG. 5 shows a summary of the effect of compound I on the cell cycle ofacute lymphoblastic leukemia (ALL) cell lines.

FIG. 6 shows a graphical summary of the effect of varying concentrationof compound I on cellular growth in breast cancer T47D and HS-578T celllines at 48 hrs and 72 hrs.

FIG. 7 shows a graphical summary of the effect of varying concentrationsof compound I on cellular growth of ovarian IGROV-1 and OVCAR-3 celllines at 48 hrs and 72 hrs.

FIG. 8 shows a summary of the effect of compound I on Aktphosphorylation in many leukemia and lymphoma cell lines.

FIG. 9 shows SDS-PAGE images and displays of Akt and pAkt in varioushematopoietic cancer cell lines as a function of the presence or absenceof compound I, showing compound I inhibits Akt phosphorylation.

FIG. 10 shows graphical summaries of apoptotic and viable cellpopulations in breast cancer cell lines as a function of varyingconcentrations of compound formula I, demonstrating that the compoundinduces apoptosis.

FIG. 11 shows the concentration of compound I in the blood of a healthyhuman subject over 12 hours after oral administration of 50, 100 and 200mg doses of said compound.

FIG. 12 shows the comparison of lesion areas in a human patientdiagnosed with mantle cell lymphoma after 28 days (1 cycle) of treatmentwith compound I and lesion areas prior to treatment.

FIG. 13 shows the ALC (absolute lymphocyte count) in the blood of apatient over a period of 4 weeks after 28 days (1 cycle) of treatmentwith the compound of formula I.

FIG. 14 shows the concentration of compound I in the blood of patientswith and without mantle cell lymphoma (MCL) over 6 hours afteradministration (50 mg BID) at day 28, compared to the concentration inthe blood of a normal healthy volunteer at day 7 (D7) using the samedosing schedule or dosing with 100 mg BID of Compound I.

FIG. 15 shows PI3K isoform expression in a panel of lymphoma andleukemia cell lines.

FIG. 16A shows cell viability and apoptosis data in leukemia cell linesexposed to Compound I. In FIG. 16B the Annexin staining indicates anincrease in apoptosis in the treated cells.

FIG. 17A-D shows PAGE results of different PI3K isoform expression inCLL patient cells.

FIG. 18A shows the induction of caspase 3 cleavage in the presence ofcompound I and FIG. 18B shows the induction of PARP cleavage in thepresence of compound I.

FIG. 19 shows increased apoptosis of Chronic Lymphocytic Leukemia (CLL)cells from poor prognosis patients caused by exposure to compound I,demonstrating that compound I is effective in drug resistant patients.

FIG. 20 shows increased apoptosis of Chronic Lymphocytic Leukemia (CLL)cells from refractory/relapsed patients caused by exposure to thecompound of formula I.

FIG. 21 shows the results of Phospho-Akt production in the absence orpresence of 0.1, 1.0, 10 μM of compound I.

FIG. 22A shows flow cytometry results relating to PI3K signaling inbasophils with no stimulation, FIG. 22B-C shows flow cytometry resultsrelating to PI3K signaling in basophils demonstrating that anti-FCεR1 orfMLP increases CD63 expression compared to no stimulation.

FIG. 23 shows inhibition of PI3K inhibition by compound I in basophils,and demonstrates that Compound I is especially effective at inhibitionof CD63 expression induced by a p110δ pathway, but also effective atmicromolar concentration to inhibit expression induced by a p110γpathway.

FIG. 24A shows pharmacokinetic data of single dose administration ofcompound I at different dose amounts in healthy volunteers, and FIG. 24Bshows a pharmacokinetic profile that maintains an effective dosage overa 12 hour period.

FIG. 25A shows the effects of various doses of compound I on glucoselevels and FIG. 25B shows the effects of various doses of compound I oninsulin levels, exhibiting little off-target activity.

FIG. 26A shows the PI3K isoform expression in a panel of DLBCL celllines.

FIG. 26 B shows an SDS-PAGE image of pAkt in DLBCL cell lines in thepresence or absence of compound I.

FIG. 27 shows the effects of a 10 μM concentration of compound I on thephosphorylation of Akt and S6 in ALL cell lines in SDS-PAGE.

FIG. 28A-B shows a dose dependent reduction of phosphorylation of Akt,S6, and GSK-3β after treatment with a series of compound I dilutions.

FIG. 29 shows dose dependent effects of compound I on ALL cell lines inthe downregulation of cFLIP, cleavage of Caspase 3, and cleavage ofPARP.

FIG. 30A shows expression of p110 delta in MM cell lines and FIG. 30Bshows expression of p110 delta in patient MM cells; and FIG. 30C showsexpression of p110 delta in MM.1S and LB cells.

FIG. 31A shows expression of p110 delta from LB and INA-6 cellstransfected with p110 delta siRNA (Si) or control siRNA (mock).

FIG. 31B shows a graph of INA-6 cell growth after transfection with p110delta siRNA (Si) or control siRNA (mock).

FIG. 31C shows the % of viable cells cultured with or without compound Ifor 48 hours.

FIG. 31D shows the % of viable MM cells after being cultured withcompound I at concentrations from 0 to 20 μM for 48 hours.

FIG. 31E shows the % of viable peripheral blood mononuclear cells fromhealthy donors after being cultured with compound I at variousconcentrations for 72 hours.

FIG. 31F shows immunoblotting results of lysates from INA-6 cellscultured with compound I (0-5 μM) for 120 hours.

FIG. 32A shows immunoblot AKT and ERK expression profiles afterculturing of INA-6 cells with compound I or LY294002 for 12 hours; FIG.32B shows INA-6 and MM.1S cells with compound I at variousconcentrations for 6 hours; FIG. 32C shows LB and INA-6 cells withcompound I for 0-6 hours.

FIG. 33A shows fluorescent and transmission electron microscopic imagesof INA-6 and LB MM cells treated with compound I for 6 hours and LC3accumulation; arrows indicate autophagosomes.

FIG. 33B shows fluorescence microscopy images of INA-6 cells treatedwith 5 μM of compound I or serum starvation for 6 hours.

FIG. 33C shows immunoblots of LC3 and beclin-1 protein levels from INA-6cells treated with or without compound I and 3-MA (3-methyl adenine, aknown inhibitor of autophagy).

FIG. 33D shows % growth of p110δ positive LB cells after treatment withup to 100 μM of 3-MA for 24 hours.

FIG. 34A-B shows the levels of growth inhibition of LB or INA-6 cellsco-cultured with 0, 5, and 10 μM of compound I in the presence orabsence of varying amounts of IL-6 or IGF-1; Legend: control media (▪);compound I at 5.0 μM (

) or 10 μM (□).

FIG. 34C and FIG. 34D show MM cell growth inhibition in the presence ofBMSC. Legend for FIG. 34C only: control media (□), Compound I 2.5 μM (

), 5 μM (

), and 10 μM (▪).

FIG. 34 E shows immunoblots of IL-6 in culture supernatants from BMSCscultured with compound I or control media for 48 hours.

FIG. 34F shows immunoblots of AKT and ERK expression profiles in INA-6cells treated with compound I cultured with our without BMSCs.

FIG. 34G shows % BMSC cell growth in two different patients afterculturing with compound I for 48 hours.

FIG. 35A shows microscopic images of HuVECs (human umbilical veinendothelial cells) cultured with 0, 1 and 10 μM of compound I for 8hours and microtubule formation assessed.

FIG. 35B shows a bar chart summarizing endothelial cell tube formationin HuVEC cells treated with compound I.

FIG. 35 C shows a graph charting % cell growth of HuVECs as a functionof the increasing culture concentration of compound I.

FIG. 35 D shows decreasing Akt and ERK expression of HuVEC cell lysatesafter being cultured with compound I for 8 hours.

FIG. 36A charts the tumor volume in SCID mice with human MM xenograftstreated with 0, 10 mg/kg or 30 mg/kg of compound II as a function oftime, showing strong in vivo activity on the human xenograft tumor.

FIG. 36 B shows a photograph comparing the tumor from human MMxenografts on a mouse treated with compound II for 12 days to a controlmouse.

FIG. 36C shows the survival rate of SCID mice with human MM xenograftstreated with 0, 10, and 30 mg/kg compound II over time.

FIG. 36D shows images from immuno-histochemistric analysis of tumorsharvested from a mouse treated with compound II in comparison to thecontrol; wherein CD31 and P-AKT positive cells are dark brown.

FIG. 36E shows immunoblots detecting PDK-1 and AKT levels from tumortissues harvested from mice treated with compound II in comparison to acontrol.

FIG. 36F shows a chart of sIL6R levels measured in mice treated with 0,10 mg/kg or 30 mg/kg of compound II over a period of 4 weeks oftreatment as determined by ELISA.

FIG. 37A show the % of viable LB or INA-6 MM cells after treatment withcompound I with varying amounts of bortezomib (B); Legend: medium (▪),compound I 1.25 μM (

), 2.5 μM (

), or 5.0 μM (□).

FIG. 37B shows immunoblots comparing levels of phosphorylation of AKT inINA-6 cells treated for 6 hours with compound I and/or bortezomib.

FIG. 38A shows PI3K isoform expression in a panel of follicular lymphomacell lines; FIG. 38B shows reduction in the expression of pAkt, Akt, pS6and S6 after exposure to compound I; and FIG. 38C shows increase in PARPand caspase-3 cleavage after exposure to compound I in a dose-dependentmanner.

FIG. 39A shows amounts of constitutive PI3K signaling in primary MCLcells in various amounts of compound I; FIG. 39B shows reduction in pAktproduction in MCL cell lines containing a survival factor and varyingamounts of compound I.

FIG. 40A-B show a computer tomography axillary image of a bulkylymphadenopathy in a patient with CLL before treatment with compound Iand after 1 cycle of treatment with compound I.

FIG. 41A shows a graph quantifying mitochondrial depolarization inducedby the BIM BH3 peptide at 0.03 μM final concentration in peripheralblood CLL cells that were BH3 profiled by FACS (n=30). FIG. 41B showsBH3 profiles from three individual patients showing pattern ofpredominant dependence on BCL2, Mcl-1, and Bcl-XL. FIG. 41C is a graphshowing BIM depolarization in treatment-naïve patients achieving apartial response (PR) or complete response (CR) by 2008 IW-CLL criteriacompared to patients with progressive disease (PD) during or within sixmonths of completing frontline CLL therapy (p=0.024). FIG. 41D is agraph showing BIM depolarization in patients with unmutated IGHV status(n=7) compared to patients with mutated IGHV status (n=18) (p=0.0026).FIG. 41E is a graph showing the correlation between percentage of VHhomology to germline with level of priming (p=0.0043).

FIG. 42A and FIG. 42B show graphs depicting CLL cell adherencequantified by whole well fluorimetry at 24 hours and 1 hour,respectively (one-tailed p=0.045 and 0.032, respectively). FIG. 42Cshows a graph depicting CLL cell viability as assessed by Annexin V/PIof PB-derived CLL cells co-cultured in the presence or absence ofStromaNKTert for 24 hours, with drug treatments as depicted in thegraph. FIG. 42D shows dose response curves for CLL cells cultured in thepresence of ABT-737 for 24 hours with or without StromaNKTert and withor without compound I. FIG. 42E shows dose response curves for CLL cellscultured in the presence of ABT-263 with or without StromaNKTert andwith or without compound I.

FIG. 43A shows aggregate CLL cell % apoptosis as measured by AnnexinV/PIfor all four patient samples. FIG. 43B shows a graph depictingmitochondrial depolarization in stroma-exposed CLL cells treated withcompound I when compared to controls (one-tailed p=0.0749). FIG. 43Cshows a graph depicting mitochondrial depolarization in stroma-exposedCLL cells treated with BAD BH3 peptide and ABT-737 with compound I whencompared to controls (one-tailed p=0.0462 and 0.0468, respectively).

MODES OF CARRYING OUT THE INVENTION

Unless otherwise defined, all terms of art, notations and otherscientific terms or terminology used herein are intended to have themeanings commonly understood by those of skill in the art to which thisinvention pertains. In some cases, terms with commonly understoodmeanings are defined herein for clarity and/or for ready reference, andthe inclusion of such definitions herein should not necessarily beconstrued to represent a substantial difference over what is generallyunderstood in the art. Many of the techniques and procedures describedor referenced herein are well understood and commonly employed usingconventional methodology by those skilled in the art. As appropriate,procedures involving the use of commercially available kits and reagentsare generally carried out in accordance with manufacturer definedprotocols and/or parameters unless otherwise noted.

The discussion of the general methods given herein is intended forillustrative purposes only. Other alternative methods and embodimentswill be apparent to those of skill in the art upon review of thisdisclosure.

A group of items linked with the conjunction “or” should not be read asrequiring mutual exclusivity among that group, but rather should also beread as “and/or” unless expressly stated otherwise. Although items,elements, or components of the invention may be described or claimed inthe singular, the plural is contemplated to be within the scope thereofunless limitation to the singular is explicitly stated.

The invention provides methods that relate to a novel therapeuticstrategy for the treatment of cancer and inflammatory diseases. In oneaspect, the invention provides a method of treating cancer or anautoimmune disease in a subject comprising administering to said subjecta compound of formula A

wherein R is H, halo, or C1-C6 alkyl; R′ is C1-C6 alkyl; or apharmaceutically acceptable salt thereof; and optionally apharmaceutically acceptable excipient.

In a particular embodiment, halo is F; and R′ is methyl, ethyl orpropyl.

In a particular embodiment, R is attached to position 5 of thequinazolinyl ring, having the structure

In a particular embodiment, R is attached to position 6 of thequinazolinyl ring, having the structure

The term ‘compound’ used herein, unless otherwise specified, refers to acompound of formula A, such as compound I, compound II, or anenantiomer, such as I″ or II″, or an enantiomeric mixture.

The “compound of formula I” or “compound I” refers to the chemicalcompound5-fluoro-3-phenyl-2-[1-(9H-purin-6-ylamino)-propyl]-3H-quinazolin-4-one,structure of formula I:

The S-enantiomer of compound I is shown here, designated I″:

The “compound of formula II” or “compound II” refers to the chemicalcompound2-(1-(9H-purin-6-ylamino)ethyl)-6-fluoro-3-phenylquinazolin-4(3H)-one,structure of formula II:

The S-enantiomer of compound II is shown here, designated II″:

In one embodiment, the compound of formula A is a compound of formula I.In another embodiment, the compound of formula A is a compound offormula II. In certain embodiments, the compound is a racemic mixture ofR- and S-enantiomers. In certain embodiments, the compound is used as amixture of enantiomers, and is often enriched with the S-enantiomer. Insome embodiments, the compound is predominantly the S-enantiomer. Insome embodiments, the compound of formula A, used in the methodsdescribed herein is at least 80% S-enantiomer. In certain embodiments,the compound is primarily composed of the S-enantiomer, wherein thecompound comprises at least 66-95%, or 85-99% of the S-enantiomer. Insome embodiments the compound has an enantiomeric excess (e.e.) of atleast 90% or at least 95% of S-enantiomer. In some embodiments thecompound has an S-enantiomeric excess (e.e.) of at least 98% or at least99%. In certain embodiments, the compound comprises at least 95% of theS-enantiomer. In the cellular and patient experiments provided in theExample section, the sample of compound I used was over 95%S-enantiomer.

In specific embodiments, the compound of formula I or II, used in themethods described herein is at least 80% S-enantiomer. In certainembodiments, the compound of formula I or II is primarily composed ofthe S-enantiomer, wherein the compound comprises at least 66-95%, or85-99% of the S-enantiomer. In some embodiments the compound of formulaI or II has an enantiomeric excess (e.e.) of at least 90% or at least95% of S-enantiomer. In some embodiments the compound of formula I or IIhas an S-enantiomeric excess (e.e.) of at least 98% or at least 99%. Incertain embodiments, the compound of formula I or II comprises at least95% of the S-enantiomer. In the cellular and patient experimentsprovided in the Example section, the sample of compound I used was over95% S-enantiomer.

In a particular embodiment, the compound selectively inhibits PI3K p110δcompared to other PI3K isoforms.

In a particular embodiment, the autoimmune disease is allergic rhinitis,asthma, COPD, or rheumatoid arthritis.

In a particular embodiment, the cancer is a hematological malignancyand/or solid tumor. In another particular embodiment, the hematologicalmalignancy is leukemia or lymphoma.

In some embodiments, lymphoma is a mature (peripheral) B-cell neoplasm.In specific embodiments, the mature B-cell neoplasm is selected from thegroup consisting of B-cell chronic lymphocytic leukemia/smalllymphocytic lymphoma; B-cell prolymphocytic leukemia; Lymphoplasmacyticlymphoma; Marginal zone lymphoma, such as Splenic marginal zone B-celllymphoma (+/−villous lymphocytes), Nodal marginal zone lymphoma(+/−monocytoid B-cells), and Extranodal marginal zone B-cell lymphoma ofmucosa-associated lymphoid tissue (MALT) type; Hairy cell leukemia;Plasma cell myeloma/plasmacytoma; Follicular lymphoma, follicle center;Mantle cell lymphoma; Diffuse large cell B-cell lymphoma (includingMediastinal large B-cell lymphoma, Intravascular large B-cell lymphoma,and Primary effusion lymphoma); and Burkitt's lymphoma/Burkitt's cellleukemia.

In some embodiments, lymphoma is selected from the group consisting ofmultiple myeloma (MM) and non-Hodgkin's lymphoma (NHL), mantle celllymphoma (MCL), follicular lymphoma, Waldenstrom's macroglobulinemia(WM) or B-cell lymphoma and diffuse large B-cell lymphoma (DLBCL).

In a further particular embodiment, leukemia is selected from the groupconsisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), and small lymphocyticlymphoma (SLL). Acute lymphocytic leukemia is also known as acutelymphoblastic leukemia and may be used interchangeably herein. Bothterms describe a type of cancer that starts from the white blood cells,lymphocytes, in the bone marrow.

In some embodiments, Non-Hodgkin's Lymphoma (NHL) falls into one of twocategories, aggressive NHL or indolent NHL. Aggressive NHL is fastgrowing and may lead to a patient's death relatively quickly. Untreatedsurvival may be measured in months or even weeks. Examples of aggressiveNHL includes B-cell neoplasms, diffuse large B-cell lymphoma, T/NK cellneoplasms, anaplastic large cell lymphoma, peripheral T-cell lymphomas,precursor B-lymphoblastic leukemia/lymphoma, precursor T-lymphoblasticleukemia/lymphoma, Burkitt's lymphoma, Adult T-cell lymphoma/leukemia(HTLV1+), primary CNS lymphoma, mantle cell lymphoma, polymorphicpost-transplantation lymphoproliferative disorder (PTLD), AIDS-relatedlymphoma, true histiocytic lymphoma, and blastic NK-cell lymphoma. Themost common type of aggressive NHL is diffuse large cell lymphoma.

Indolent NHL is slow growing and does not display obvious symptoms formost patients until the disease has progressed to an advanced stage.Untreated survival of patients with indolent NHL may be measured inyears. Non-limiting examples include follicular lymphoma, smalllymphocytic lymphoma, marginal zone lymphoma (such as extranodalmarginal zone lymphoma (also called mucosa associated lymphoidtissue—MALT lymphoma), nodal marginal zone B-cell lymphoma (monocytoidB-cell lymphoma), splenic marginal zone lymphoma), and lymphoplasmacyticlymphoma (Waldenstrom's macroglobulinemia).

In some cases, histologic transformation may occur, e.g., indolent NHLin patients may convert to aggressive NHL.

In some embodiments, the invention provides methods of treating apatient with aggressive NHL or indolent NHL.

In some embodiments, the invention provides methods of treating apatient with a condition selected from the group consisting of mantlecell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicularlymphoma (FL), acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), and small lymphocyticlymphoma (SLL), multiple myeloma (MM), and marginal zone lymphoma.

In some embodiments, the methods of the invention are administered topatients with relapsed or refractory conditions.

In another embodiment, the cancer is breast, lung, colon or prostatecancer.

In a particular embodiment, the cancer or autoimmune disease isassociated with abnormal PI3K activity compared to PI3K activity in asubject without cancer or without an autoimmune disease.

In a particular embodiment, the preferred subject is refractory tochemotherapy treatment, or in relapse after treatment with chemotherapy.In an alternative embodiment, the subject is a de novo patient.

In a particular embodiment, the method comprises reducing the level ofPI3Kδ activity in said patient.

In a particular embodiment, the subject is a human subject.

Subjects that undergo treatment with known therapeutic agents mayexperience resistance to treatment. For example, although bortezomib wasFDA approved for relapsed/refractory, relapsed, and newly diagnosed MM,some patients do not respond and others acquire resistance tobortezomib. In some embodiments, the quinazolinone compound describedherein synergistically augments efficacy of a known therapeutic agent.In some embodiments, the compounds described herein can augment any ofthe therapeutic agents described herein. In more specific embodiments,the compounds described herein synergistically augment proteasomeinhibitors. In some of the foregoing embodiments, the subject isresistant to chemotherapeutic treatment. In some of the foregoingembodiments, the subject is resistant to proteasome inhibitors. In someof the foregoing embodiments, the subject is resistant bortezomib orcarfilzomib. In one example, the compounds described hereinsynergistically augment bortezomib-induced MM cytotoxicity. Withoutbeing bound by theory, in some embodiments, the compounds discussedherein inhibit bortezomib-induced phosphorylation of AKT. In someembodiments, the methods described herein are used to overcomeresistance to proteasome inhibitor treatment. In some embodiments, theinvention provides a method to treat a subject that is resistant or hasdeveloped a resistance to therapeutic agents.

While not being bound by theory, the synergistic effects between acompound of formula A and conventional therapies may be attributed tothe ability of the compound of the invention to induce tumor cellmobilization into peripheral circulation. Inducing the peripheralcirculation of the tumor cells increases the ability of conventionaltherapy to act upon and more effectively neutralize the tumor. Thissynergy has been demonstrated in CLL patients.

Accordingly, the method comprises administering in addition to acompound of formula A to a patient, a therapeutically effective amountof at least one additional therapeutic agent and/or a therapeuticprocedure selected to treat said cancer or autoimmune disease in saidpatient. “Therapeutic agent” may refer to one or more compounds, as usedherein. The therapeutic agent may be a standard or experimentalchemotherapy drug. The therapeutic agent may comprise a combination ofmore than one chemotherapy drug. Typical chemotherapy drug combinationsare listed a-q herein. A particular therapeutic agent may be chosendepending on the type of disease being treated. Non-limiting examples ofconventional chemotherapeutic treatments for particular hematologicdisease are described in later sections. In a particular embodiment, theinvention provides a method to treat a hematopoietic cancer patient,e.g., a CLL patient, with bortezomib and a compound of formula A (e.g.,formula I, II, I″, or II″), wherein the combination provides asynergistic effect.

In a particular embodiment, said therapeutic agent is selected from thefollowing group consisting of bortezomib (Velcade®), carfilzomib(PR-171), PR-047, disulfiram, lactacystin, PS-519, eponemycin,epoxomycin, aclacinomycin, CEP-1612, MG-132, CVT-63417, PS-341, vinylsulfone tripeptide inhibitors, ritonavir, PI-083,(+/−)-7-methylomuralide, (−)-7-methylomuralide, perifosine, rituximab,sildenafil citrate (Viagra®), CC-5103, thalidomide, epratuzumab(hLL2-anti-CD22 humanized antibody), simvastatin, enzastaurin,Campath-1H®, dexamethasone, DT PACE, oblimersen, antineoplaston A10,antineoplaston AS2-1, alemtuzumab, beta alethine, cyclophosphamide,doxorubicin hydrochloride, PEGylated liposomal doxorubicinhydrochloride, prednisone, prednisolone, cladribine, vincristinesulfate, fludarabine, filgrastim, melphalan, recombinant interferonalfa, carmustine, cisplatin, cyclophosphamide, cytarabine, etoposide,melphalan, dolastatin 10, indium In 111 monoclonal antibody MN-14,yttrium Y 90 humanized epratuzumab, anti-thymocyte globulin, busulfan,cyclosporine, methotrexate, mycophenolate mofetil, therapeuticallogeneic lymphocytes, Yttrium Y 90 ibritumomab tiuxetan, sirolimus,tacrolimus, carboplatin, thiotepa, paclitaxel, aldesleukin, recombinantinterferon alfa, docetaxel, ifosfamide, mesna, recombinantinterleukin-12, recombinant interleukin-11, Bcl-2 family proteininhibitor ABT-263, denileukin diftitox, tanespimycin, everolimus,pegfilgrastim, vorinostat, alvocidib, recombinant flt3 ligand,recombinant human thrombopoietin, lymphokine-activated killer cells,amifostine trihydrate, aminocamptothecin, irinotecan hydrochloride,caspofungin acetate, clofarabine, epoetin alfa, nelarabine, pentostatin,sargramostim, vinorelbine ditartrate, WT-1 analog peptide vaccine, WT1126-134 peptide vaccine, fenretinide, ixabepilone, oxaliplatin,monoclonal antibody CD19, monoclonal antibody CD20, omega-3 fatty acids,mitoxantrone hydrochloride, octreotide acetate, tositumomab and iodine I131 tositumomab, motexafin gadolinium, arsenic trioxide, tipifarnib,autologous human tumor-derived HSPPC-96, veltuzumab, bryostatin 1,anti-CD20 monoclonal antibodies, chlorambucil, pentostatin, lumiliximab,apolizumab, Anti-CD40, and ofatumumab, or a combination thereof.Combinations of therapeutic agents are used in current and experimentaltherapies such as those combinations a-q listed above.

In some embodiments, the therapeutic agent is preferably a proteasomeinhibitor. In some embodiments, the methods comprise administering acompound with a proteasome inhibitor. Proteasome inhibitors includenatural and synthetic compounds. Non-limiting examples of proteasomeinhibitors include bortezomib,([(1R)-3-methyl-1-({(2S)-3-phenyl-2-[(pyrazin-2-ylcarbonyl)amino]propanoyl}amino)butyl]boronicacid), which is marketed as ‘Velcade®’ by Millennium pharmaceuticals;carfilzomib (PR-171) and the oral analog, PR-047, both of which aredeveloped by Proteolix, Inc. Other examples of proteasome inhibitorsinclude disulfiram; lactacystin; synthetic compounds such as PS-519,eponemycin, epoxomycin, and aclacinomycin; calpain inhibitors, such asCEP-1612, MG-132, CVT-63417, PS-341; vinyl sulfone tripeptideinhibitors; ritonavir; PI-083; (+/−)-7-methylomuralide; and(−)-7-methylomuralide. In particular embodiments, the compound offormula A is administered in combination with bortezomib or carfilzomib.In more particular embodiments, the compound of formula I isadministered in combination with bortezomib or carfilzomib. In otherparticular embodiments, the compound of formula II is administered incombination with bortezomib or carfilzomib. In particular embodiments,the compound of formula A is administered in combination with rituximabor ofatumumab. In more particular embodiments, the compound of formula Iis administered in combination with rituximab or ofatumumab. In otherparticular embodiments, the compound of formula II is administered incombination with rituximab or ofatumumab.

In one aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula I:

or a pharmaceutically acceptable salt thereof; and at least onepharmaceutically acceptable excipient. In one embodiment, thecomposition is enriched with the S-enantiomer.

In another aspect, the invention provides a pharmaceutical compositioncomprising a compound of Formula II:

or a pharmaceutically acceptable salt thereof; and at least onepharmaceutically acceptable excipient. In one embodiment, thecomposition is enriched with the S-enantiomer.

In one aspect, the invention provides a method of treating multiplemyeloma (MM) in a patient comprising administering a combination of acompound of formula A and an additional therapeutic agent. In someembodiments, formula A is compound I or II. In specific embodiments,formula A is compound I″. In other embodiments, formula A is compoundII″. In some of the foregoing embodiments the additional therapeuticagent is a proteasome inhibitor. In specific embodiments the additionaltherapeutic agent is bortezomib. In a specific embodiment, the method oftreating multiple myeloma in a patient comprises administering compoundI″ with bortezomib. In a specific embodiment, the method of treatingmultiple myeloma in a patient comprises administering compound II″ withbortezomib. In some of the foregoing embodiments, compound I″ or II″ hasan enantiomeric excess of at least 60%. In some of the foregoingembodiments, compound I″ or II″ has an enantiomeric excess of at least70%. In some of the foregoing embodiments, compound I″ or II″ has anenantiomeric excess of at least 80%. In some of the foregoingembodiments, compound I″ or II″ has an enantiomeric excess of at least90%. In some of the foregoing embodiments, compound I″ or II″ has anenantiomeric excess of at least 95%. In some of the foregoingembodiments, compound I″ or II″ has an enantiomeric excess of at least98%. In some of the foregoing embodiments, compound I″ or II″ has anenantiomeric excess of at least 99%.

In a particular embodiment, a combination of therapeutic agents isadministered with a compound of Formula A, wherein said combination isselected from the group consisting of

a) CHOP (cyclophosphamide, doxorubicin, vincristine, prednisone);

b) R-CHOP (rituximab-CHOP);

c) hyperCVAD (hyperfractionated cyclophosphamide, vincristine,doxorubicin, dexamethasone, methotrexate, cytarabine);

d) R-hyperCVAD (rituximab-hyperCVAD);

e) FCM (fludarabine, cyclophosphamide, mitoxantrone);

f) R-FCM (rituximab, fludarabine, cyclophosphamide, mitoxantrone);

g) bortezomib and rituximab;

h) temsirolimus and rituximab;

i) temsirolimus and Velcade®;

j) Iodine-131 tositumomab (Bexxar®) and CHOP;

k) CVP (cyclophosphamide, vincristine, prednisone);

l) R-CVP (rituximab-CVP);

m) ICE (iphosphamide, carboplatin, etoposide);

n) R-ICE (rituximab-ICE);

o) FCR (fludarabine, cyclophosphamide, rituximab);

p) FR (fludarabine, rituximab); and

q) D.T. PACE (dexamethasone, thalidomide, cisplatin, Adriamycin®,cyclophosphamide, etoposide).

In alternative embodiments, the compound is used in combination with atherapeutic procedure. In a particular embodiment, the therapeuticprocedure is selected from the group consisting of peripheral blood stemcell transplantation, autologous hematopoietic stem celltransplantation, autologous bone marrow transplantation, antibodytherapy, biological therapy, enzyme inhibitor therapy, total bodyirradiation, infusion of stem cells, bone marrow ablation with stem cellsupport, in vitro-treated peripheral blood stem cell transplantation,umbilical cord blood transplantation, immunoenzyme technique,immunohistochemistry staining method, pharmacological study, low-LETcobalt-60 gamma ray therapy, bleomycin, conventional surgery, radiationtherapy, high-dose chemotherapy and nonmyeloablative allogeneichematopoietic stem cell transplantation.

In a particular embodiment, the method further comprises obtaining abiological sample from said patient; and analyzing said biologicalsample with an analytical procedure selected from the group consistingof blood chemistry analysis, chromosomal translocation analysis, needlebiopsy, fluorescence in situ hybridization, laboratory biomarkeranalysis, immunohistochemistry staining method, flow cytometry or acombination thereof.

For nomenclature purposes, the quinazolinyl and purinyl components ofthe compound are numbered accordingly:

As used herein, the term “alkyl,” includes straight-chain,branched-chain and cyclic monovalent hydrocarbyl radicals, andcombinations of these, which contain only C and H when they areunsubstituted. Examples include methyl, ethyl, isobutyl, cyclohexyl,cyclopentylethyl, and the like. The total number of carbon atoms in eachsuch group is sometimes described herein, e.g., when the group cancontain up to ten carbon atoms it can be represented as 1-10C or asC1-C10 or C1-10.

“Halo”, as used herein, includes fluoro, chloro, bromo and iodo. Fluoroand chloro are often preferred.

The term “selective PI3Kδ inhibitor” or “selective PI3Kβ inhibitor”,etc., as used herein, refers to a compound that inhibits the PI3Kδ orPI3Kβ isozyme, respectively, more effectively than at least one otherisozymes of the PI3K family. The selective inhibitor may also be activeagainst other isozymes of PI3K, but requires higher concentrations toachieve the same degree of inhibition of the other isozymes. “Selective”can also be used to describe a compound that inhibits a particularPI3-kinase more so than a comparable compound. A “selective PI3Kδinhibitor” compound is understood to be more selective for PI3Kδ thancompounds conventionally and generically designated PI3K inhibitors,e.g., wortmannin or LY294002. Concomitantly, wortmannin and LY294002 aredeemed “nonselective PI3K inhibitors.” In certain embodiments, compoundsof any type that selectively negatively regulate PI3Kδ expression oractivity can be used as selective PI3Kδ inhibitors in the methods of theinvention. Moreover, compounds of any type that selectively negativelyregulate PI3Kδ expression or activity and that possess acceptablepharmacological properties can be used as selective PI3Kδ inhibitors inthe therapeutic methods of the invention. Without being bound by theory,targeting p110 delta inhibition with a compound of the inventionprovides a novel approach for the treatment of hematologicalmalignancies because this method inhibits constitutive signalingresulting in direct destruction of the tumor cell. In addition, withoutbeing bound by theory, p110 delta inhibition repressesmicroenvironmental signals which are crucial for tumor cell homing,survival and proliferation.

In an alternative embodiment, compounds of any type that selectivelynegatively regulate PI3Kβ expression or activity can be used asselective PI3Kβ inhibitors in the methods of the invention. Moreover,compounds of any type that selectively negatively regulate PI3Kβexpression or activity and that possess acceptable pharmacologicalproperties can be used as selective PI3Kβ inhibitors in the therapeuticmethods of the invention.

“Treating” as used herein refers to inhibiting a disorder, i.e.,arresting its development; relieving the disorder, i.e., causing itsregression; or ameliorating the disorder, i.e., reducing the severity ofat least one of the symptoms associated with the disorder. In someembodiments, “treating” refers to preventing a disorder from occurringin an animal that can be predisposed to the disorder, but has not yetbeen diagnosed as having it. “Disorder” is intended to encompass medicaldisorders, diseases, conditions, syndromes, and the like, withoutlimitation.

“Autoimmune disease” as used herein refers to any group of disorders inwhich tissue injury is associated with humoral or cell-mediatedresponses to the body's own constituents.

In another aspect, the invention includes a method for suppressing afunction of basophils and/or mast cells, and thereby enabling treatmentof diseases or disorders characterized by excessive or undesirablebasophil and/or mast cell activity. According to the method, a compoundof the invention can be used that selectively inhibits the expression oractivity of phosphatidylinositol 3-kinase delta (PI3Kδ) in the basophilsand/or mast cells. Preferably, the method employs a PI3Kδ inhibitor inan amount sufficient to inhibit stimulated histamine release by thebasophils and/or mast cells. Accordingly, the use of such compounds andother PI3Kδ selective inhibitors can be of value in treating diseasescharacterized by histamine release, i.e., allergic disorders, includingdisorders such as chronic obstructive pulmonary disease (COPD), asthma,ARDS, emphysema, and related disorders.

The present invention enables methods of treating such diseases asarthritic diseases, such as rheumatoid arthritis, psoriatic arthritis,monoarticular arthritis, osteoarthritis, gouty arthritis, spondylitis;Behçet disease; sepsis, septic shock, endotoxic shock, gram negativesepsis, gram positive sepsis, and toxic shock syndrome; multiple organinjury syndrome secondary to septicemia, trauma, or hemorrhage;ophthalmic disorders such as allergic conjunctivitis, vernalconjunctivitis, uveitis, and thyroid-associated ophthalmopathy;eosinophilic granuloma; pulmonary or respiratory disorders such asasthma, chronic bronchitis, allergic rhinitis, ARDS, chronic pulmonaryinflammatory disease (e.g., chronic obstructive pulmonary disease),silicosis, pulmonary sarcoidosis, pleurisy, alveolitis, vasculitis,emphysema, pneumonia, bronchiectasis, and pulmonary oxygen toxicity;reperfusion injury of the myocardium, brain, or extremities; fibrosissuch as cystic fibrosis; keloid formation or scar tissue formation;atherosclerosis; autoimmune diseases, such as systemic lupuserythematosus (SLE), autoimmune thyroiditis, multiple sclerosis, someforms of diabetes, and Reynaud's syndrome; and transplant rejectiondisorders such as graft-versus-host disease (GVHD) and allograftrejection; chronic glomerulonephritis; inflammatory bowel diseases suchas chronic inflammatory bowel disease (CIBD), Crohn's disease,ulcerative colitis, and necrotizing enterocolitis; inflammatorydermatoses such as contact dermatitis, atopic dermatitis, psoriasis, orurticaria; fever and myalgias due to infection; central or peripheralnervous system inflammatory disorders such as meningitis, encephalitis,and brain or spinal cord injury due to minor trauma; Sjogren's syndrome;diseases involving leukocyte diapedesis; alcoholic hepatitis; bacterialpneumonia; antigen-antibody complex mediated diseases; hypovolemicshock; Type I diabetes mellitus; acute and delayed hypersensitivity;disease states due to leukocyte dyscrasia and metastasis; thermalinjury; granulocyte transfusion-associated syndromes; andcytokine-induced toxicity.

The method can have utility in treating subjects who are or can besubject to reperfusion injury, i.e., injury resulting from situations inwhich a tissue or organ experiences a period of ischemia followed byreperfusion. The term “ischemia” refers to localized tissue anemia dueto obstruction of the inflow of arterial blood. Transient ischemiafollowed by reperfusion characteristically results in neutrophilactivation and transmigration through the endothelium of the bloodvessels in the affected area. Accumulation of activated neutrophils inturn results in generation of reactive oxygen metabolites, which damagecomponents of the involved tissue or organ. This phenomenon of“reperfusion injury” is commonly associated with conditions such asvascular stroke (including global and focal ischemia), hemorrhagicshock, myocardial ischemia or infarction, organ transplantation, andcerebral vasospasm. To illustrate, reperfusion injury occurs at thetermination of cardiac bypass procedures or during cardiac arrest whenthe heart, once prevented from receiving blood, begins to reperfuse. Itis expected that inhibition of PI3Kδ activity will result in reducedamounts of reperfusion injury in such situations.

In certain embodiments, the invention provides methods to treat a solidtumor. In specific embodiments, the cancer is breast, lung, colon, orprostate cancer. In certain embodiments, the invention provides methodsto treat a solid tumor that is associated with abnormal or undesirablecellular signaling activity mediated by PI3Kβ. In certain embodiments, asolid tumor is selected from the group consisting of pancreatic cancer;bladder cancer; colorectal cancer; breast cancer, including metastaticbreast cancer; prostate cancer, including androgen-dependent andandrogen-independent prostate cancer; renal cancer, including, e.g.,metastatic renal cell carcinoma; hepatocellular cancer; lung cancer,including, e.g., non-small cell lung cancer (NSCLC), bronchioloalveolarcarcinoma (BAC), and adenocarcinoma of the lung; ovarian cancer,including, e.g., progressive epithelial or primary peritoneal cancer;cervical cancer; gastric cancer; esophageal cancer; head and neckcancer, including, e.g., squamous cell carcinoma of the head and neck;melanoma; neuroendocrine cancer, including metastatic neuroendocrinetumors; brain tumors, including, e.g., glioma, anaplasticoligodendroglioma, adult glioblastoma multiforme, and adult anaplasticastrocytoma; bone cancer; and soft tissue sarcoma.

Genetic ablation of p110δ has been found to result in mild phenotyperestricted to immune system. General observations include organisms thatare fertile with no gross anatomical or behavioral abnormalities. Ahistological examination revealed major organs to appear normal. Thetotal class I PI3K activity was reduced 30-50% in B and T cells. Inaddition, no increase in susceptibility to infections was observed.Furthermore, the effect on the hematopoietic system includes normalperipheral blood cell counts, the occurrence of lymphoid hypoplasia andthe lack of germinal centers in spleen and lymph nodes, a reduced numberof B220+IgM+B cell progenitors in bone marrow, a reduced level of serumimmunoglobulin, and normal T cell development in the thymus.

Genetic ablation of p110δ affects myeloid and B cell signaling, which isimportant for oncogenesis. In particular, tyrosine kinase signaling,development, proliferation and survival are affected in myeloid cells. Bcell function is most affected and includes proliferation,differentiation, apoptosis, and response to B cell survival factors(BCR, CD40, IL-4, chemokines). Thus, the invention includes methods oftreating disease states in which one or more of these myeloid and B cellfunctions are abnormal or undesirable.

A pan PI3K inhibitor that targets on a molecular level p110α, p110β,p110α, p110γ, (hvPS34, mTOR, DNA-PK, and others), in turn targets alltissues. The potential clinical indication includes cancer but clinicaladverse events include hyperinsulinemia in cancer patients. Theadvantage of a p110δ selective inhibitor which targets cells mediatinginflammation and cancer cells, wherein potential clinical indicationinclude cancer, rheumatoid arthritis, asthma, allergies and COPD, isthat treatment is well tolerated, and side effects like hyperinsulinemiaare avoided. Thus in one aspect the invention provides a method to treatpatients having insulin resistance, or type 2 diabetes, for cancer,rheumatoid arthritis, asthma, allergies, COPD, or other conditionstreatable with the compounds of the invention. For patients needing suchtreatment who have excessive insulin conditions or tendencies, thecompounds of the invention are particularly advantageous over pan-PI3Kinhibitors. In certain embodiments, a compound of formula I or I″ ispreferred because it provides therapeutic benefits to treatinghematologic malignancies without adversely affecting insulin signaling.

In one embodiment, the invention relates to methods of inhibiting PI3Kp110β. In another embodiment, the invention relates to methods ofinhibiting PI3K p110β or p110γ.

In certain embodiments, the method described herein has little or no offtarget activity. In particular, compound of formula I used in the methodshow little activity against over 300 protein kinases including thosesummarized in Table 3 of Example 16. In certain embodiments, the methoddescribed herein has no or minimal hyperinsulinemia effects in cancerpatients compared to methods comprising the administration of pan-PI3Kinhibitors. In certain embodiments, the method described herein isuseful in targeting cells mediating Akt phosphorylation, because thecompounds of Formula A inhibit Akt phosphorylation. Suitable patientsfor treatment with the compounds of the invention can thus be selected,in one embodiment, by selecting a patient exhibiting elevated Aktphosphorylation associated with a hematopoietic cancer such as lymphoma,leukemia or multiple myeloma.

The methods herein avoid off-target liabilities and are characterized bynegative results in receptor gram screens, having no hERG inhibition andno significant P450 inhibition.

Another advantage of the inventive method is the absence of adversecardiovascular, respiratory, or central nervous system effects asdemonstrated in safety pharmacology studies. In addition, a 28-daytoxicity study in rats and dogs demonstrated a high therapeutic index,e.g., a NOAEL (no observable adverse effect level)>>10 μM. This is thehighest experimental dose of a chemical at which there is nostatistically or biologically significant increase in frequency orseverity of a toxicological effect between an exposed group and itsappropriate control. Adverse effects are defined as any effects thatresult in functional impairment and/or pathological lesions that mayaffect the performance of the whole organism or that reduce anorganism's ability to respond to an additional challenge.

In another embodiment, the inventive methods are non-genotoxic in astandard battery of tests.

Another advantage of the invention is that compound selectivity for oneor two PI3K isoforms results in an improved safety profile overcompounds having pan-PI3K inhibition. In yet another advantage, compoundI has a favorable pharmacokinetic profile with good target coverage, andno adverse effects on glucose or insulin levels, and is well toleratedat doses above commonly used therapeutic doses by normal healthyvolunteers. Another advantage of the invention includes the ability totreat a wide range of hematological malignancies as demonstrated by theexamples herein.

In certain embodiments, the methods of the invention are directedtowards treating a cancer or an autoimmune disease. In certainembodiments, the cancer is a hematological malignancy. In specificembodiments, the hematological malignancy is selected from the groupconsisting of acute lymphocytic leukemia (ALL), acute myeloid leukemia(AML), chronic lymphocytic leukemia (CLL), multiple myeloma (MM), andnon-Hodgkin lymphoma (NHL). In certain embodiments, the non-Hodgkinlymphoma is selected from the group consisting of large diffuse B-celllymphoma (LDBCL), mantle cell lymphoma (MCL), Waldenstrom'smacroglobulinemia (WM) and lymphoplasmacytic lymphoma.

PI3K is implicated in many hematological malignancies and preclinicalproof of concept relating to treatment with compound I has beenestablished. The table below summarizes particular hematologicalmalignancies and the method of action on the primary patient cell ordisease cell line.

Effects of compounds Indication of formula A Chronic LymphocyticLeukemia Primary patient cells (CLL) Induces apoptosis Blocks survivalfactors Acute Myelogenous Leukemia (AML) Primary patient cells BlocksPI3K signaling Inhibits proliferation Acute Lymphocytic Leukemia (ALL)Cell Lines Blocks PI3K signaling Induces apoptosis Non-Hodgkin'sLymphomas (NHL) Cell Lines (MCL, DLBCL, FL) Blocks PI3K signalingInduces apoptosis Multiple Myeloma (MM) Primary patient cells P110 δoverexpressed in 24/24 samples Induces apoptosis

Data provided herein demonstrates that the compounds of the inventionare useful to treat lymphomas and leukemias. Lymphomas and leukemiasgenerally express the delta isoform of p110 selectively, e.g., FIG. 15demonstrates that p110δ is prevalent in most lymphoma cell lines, whilep110α is not generally observed. Moreover, data presented in FIG. 16Ashows that cell cultures from six different leukemia cell lines weresensitive to Compound I, and were strongly affected by 5-10 micromolarconcentrations of this compound. FIGS. 8 and 9 support compound I asreducing Akt(Ser473) production in several cell lines.

CLL, for example, produces mainly p110δ and to a lesser extent p110γ forsignaling purposes, thus compounds that inhibit p110δ and/or p110γ areexpected to exhibit selective cytotoxicity towards these cells. InExample 3, for example, shows dose-dependent cytotoxicity for compound I(FIG. 3), in CLL cells, including cells taken from poor prognosispatients (FIG. 19), and cells from patients shown to be resistant toother CLL treatments (FIG. 20). In addition, Example 13 and FIG. 13demonstrate that compound I administered to a CLL patient at a rate of50 mg BID for a 28-day cycle provides a significant therapeutic effect.An ALC concentration percent decrease in lymphocytes is observed. Thusin one aspect, the invention provides methods for treating CLL patientswith drug-resistant CLL using compounds of Formula A. On the other hand,Example 17 suggests that a fibroblast cell line relying mainly on p110αfor signaling was not sensitive to Compound I. Thus in one aspect,patient selection can include excluding patients having a cancer thatrelies mainly on p110α for signaling.

The compounds of Formula A are also useful to treat lymphoma, includingboth B-cell and T-cell lymphomas. Data in FIG. 4 demonstrates that sixdifferent ALL cell lines were sensitive to Compound I, which caused asignificant reduction in cell viability in all six cell lines.

FIG. 12 and Example 12 demonstrate that mantle cell lymphoma patientstreated with 50 mg BID of Compound I for 28 days experienced on averagea 44% decrease in tumor burden. Moreover, FIG. 14 demonstrates that anMCL patient at the end of the 28 day cycle experienced similar plasmalevels of Compound I following administration of a 50 mg dose to thatobserved in a normal healthy volunteer (NHV); thus the compound does notbuild up excessively over the course of a cycle of treatment, nor doesthe patient become tolerant by increased metabolism over the course of atreatment cycle.

In addition, the compounds of Formula A, or Formula I, are useful totreat hematopoietic cancers that constitutively express Aktphosphorylation activity. Example 8, and FIGS. 8 and 9 list cancer celllines that demonstrate constitutive Akt phosphorylation, includingB-cell lymphomas, T-cell lymphomas, ALL, malignant histiocytosis, DLBCLand AML. Exposure of the cell to compound I results in the reduction ofAkt phosphorylation. See also Example 19, which shows that constitutiveAkt phosphorylation was inhibited by Compound I in 13 of 13 cell lines.

In certain embodiments, the cancer is a solid tumor. In specificembodiments, the cancer is breast, ovarian, lung, colon, or prostatecancer. FIG. 6, for example, shows that Compound I reduces cellularproliferation of two breast cancer cell lines, and FIG. 10 illustratescytotoxicity to three different breast cancer cell lines. Similarly,FIG. 7 demonstrates that Compound I is cytotoxic to two ovarian cancercell lines.

For the treatment of a solid tumor, it is advantageous to use a compoundof Formula A that expresses good activity (e.g., IC₅₀ less than about 1μM, and preferably less than about 250 nM—see Example 15) against p110β,since solid tumors often utilize this isozyme rather than or more thanp110δ. Thus a compound of formula A that has an IC₅₀ less than about 250nM is preferred for treatment of a solid tumor; compound I, I″, II, orII″ is suitable for this use, as demonstrated herein.

In some embodiments, the subject for treatments described herein as onewho has been diagnosed with at least one of the conditions describedherein as treatable by the use of a compound of Formula A. In someembodiments, the subject has been diagnosed with a cancer named herein,and has proven refractory to treatment with at least one conventionalchemotherapeutic agent. For instance, patients who have failed torespond to treatments such as proteasome inhibitors, autologous stemcell transplant, CHOP regimens, rituximab, fludarabine, alemtuzumab,conventional anticancer nucleoside analogues and alkylating agentsfrequently respond to the methods of treatment described herein. Thus,in one embodiment, the treatments of the invention are directed topatients who have received one or more than one such treatment.

In certain embodiments, the autoimmune disease is allergic rhinitis,asthma, chronic obstructive pulmonary disease (COPD), or rheumatoidarthritis.

In certain embodiments, the methods of the invention are directed toB-cell, or B lymphocyte, related diseases. B-cells play a role in thepathogenesis of autoimmune diseases.

The compounds of Formula A (particularly Formulas I, I″, II and II″) aresuitable for treating a variety of subjects having the conditionsdescribed herein, especially hematological cancers in humans. In someembodiments, the subject selected for treatment of a hematologicalmalignancy that is a subject experiencing relapse after other treatmentsor is refractory to other treatments. In some embodiments, the subjectis selected for treatment of a hematological malignancy that isresistant to other cancer drugs. In some embodiments, the subject isselected for treatment of a hematological malignancy that exhibits ahigh level of p110δ activity. In some embodiments, the subject isselected for treatment of a hematological malignancy that exhibits arelatively low level of p110α activity. In some embodiments, the subjectis selected for treatment of a hematological malignancy thatconstitutively expresses Akt phosphorylation activity.

In one embodiment, the method described herein comprises administeringto a subject a compound of formula A described herein, in combinationwith a therapy used to treat cancer or an autoimmune disease. “Therapy”or “treatment”, as used herein, is a treatment of cancer or anautoimmune disease by any well-known conventional or experimental formof treatment used to treat cancer or an autoimmune disease that does notinclude the use of a compound of formula A. In certain embodiments, thecombination of a compound of formula A with a conventional orexperimental therapy used to treat cancer or an autoimmune diseaseprovides beneficial and/or desirable treatment results superior toresults obtained by treatment without the combination. In certainembodiments, therapies used to treat cancer or an autoimmune disease arewell-known to a person having ordinary skill in the art and aredescribed in the literature. Therapies include, but are not limited to,chemotherapy, combinations of chemotherapy, biological therapies,immunotherapy, radioimmunotherapy, and the use of monoclonal antibodies,and vaccines.

In some of the foregoing embodiments, the combination method providesfor a compound of formula A administered simultaneously with or duringthe period of administration of the therapy. In some of the foregoingembodiments the compound of formula A is administered simultaneouslywith the other chemotherapeutic treatment. In certain embodiments, thecombination method provides for a compound of formula A administeredprior to or after the administration of the therapy.

In some of the foregoing embodiments, the subject is refractory to atleast one standard or experimental chemotherapy. In some of theforegoing embodiments, the subject is refractory to at least twostandard or experimental chemotherapies. In some of the foregoingembodiments, the subject is refractory to at least three standard orexperimental chemotherapies. In some of the foregoing embodiments, thesubject is refractory to at least four standard or experimentalchemotherapies.

In some of the foregoing embodiments, the subject is refractory to atleast one standard or experimental chemotherapy selected from the groupconsisting of fludarabine, rituximab, alkylating agents, alemtuzumab andthe chemotherapy treatments a-q listed above.

In some of the foregoing embodiments, the subject is refractory to atleast two standard or experimental chemotherapies selected from thegroup consisting of fludarabine, rituximab, alkylating agents,alemtuzumab and the chemotherapy treatments a-q listed above.

In some of the foregoing embodiments, the subject is refractory to atleast three standard or experimental chemotherapies selected from thegroup consisting of fludarabine, rituximab, alkylating agents,alemtuzumab and the chemotherapy treatments a-q listed above.

In some of the foregoing embodiments, the subject is refractory to atleast four standard or experimental chemotherapies selected from thegroup consisting of fludarabine, rituximab, alkylating agents,alemtuzumab and the chemotherapy treatments a-q listed above.

The exact details regarding the administration of the combination may bedetermined experimentally. The refinement of sequence and timing ofadministering a compound of formula A with a selected therapy will betailored to the individual subject, the nature of the condition to betreated in the subject, and generally, the judgment of the attendingpractitioner.

Non-limiting examples of experimental or standard therapy are describedbelow. In addition, treatment of certain lymphomas is reviewed inCheson, B. D., Leonard, J. P., “Monoclonal Antibody Therapy for B-CellNon-Hodgkin's Lymphoma” The New England Journal of Medicine 2008,359(6), p. 613-626; and Wierda, W. G., “Current and InvestigationalTherapies for Patients with CLL” Hematology 2006, p. 285-294. Lymphomaincidence patterns in the United States is profiled in Morton, L. M., etal. “Lymphoma Incidence Patterns by WHO Subtype in the United States,1992-2001” Blood 2006, 107(1), p. 265-276.

Treatment of non-Hodgkin's lymphomas, especially of B cell origin,include, but are not limited to use of monoclonal antibodies, standardchemotherapy approaches (e.g., CHOP, CVP, FCM, MCP, and the like),radioimmunotherapy, and combinations thereof, especially integration ofan antibody therapy with chemotherapy.

Non-limiting examples of unconjugated monoclonal antibodies forNon-Hodgkin's lymphoma/B-cell cancers include rituximab, alemtuzumab,human or humanized anti-CD20 antibodies, lumiliximab, anti-TRAIL,bevacizumab, galiximab, epratuzumab, SGN-40, and anti-CD74. Non-limitingexamples of experimental antibody agents used in treatment ofNon-Hodgkin's lymphoma/B-cell cancers include ofatumumab, ha20,PRO131921, alemtuzumab, galiximab, SGN-40, CHIR-12.12, epratuzumab,lumiliximab, apolizumab, milatuzumab, and bevacizumab. Any of themonoclonal antibodies can be combined with rituximab, fludarabine, or achemotherapy agent/regimen.

Non-limiting examples of standard regimens of chemotherapy forNon-Hodgkin's lymphoma/B-cell cancers include CHOP (cyclophosphamide,doxorubicin, vincristine, prednisone), FCM (fludarabine,cyclophosphamide, mitoxantrone), CVP (cyclophosphamide, vincristine andprednisone), MCP (mitoxantrone, chlorambucil, and prednisolone), R-CHOP(rituximab plus CHOP), R-FCM (rituximab plus FCM), R-CVP (rituximab plusCVP), and R-MCP (R-MCP).

Non-limiting examples of radioimmunotherapy for Non-Hodgkin'slymphoma/B-cell cancers include yttrium-90-labeled ibritumomab tiuxetan,and iodine-131-labeled tositumomab. These therapeutic agents areapproved for use in subjects with relapsed or refractory follicular orlow-grade lymphoma.

Therapeutic treatments for mantle cell lymphoma include combinationchemotherapies such as CHOP (cyclophosphamide, doxorubicin, vincristine,prednisone), hyperCVAD (hyperfractionated cyclophosphamide, vincristine,doxorubicin, dexamethasone, methotrexate, cytarabine) and FCM(fludarabine, cyclophosphamide, mitoxantrone). In addition, theseregimens can be supplemented with the monoclonal antibody rituximab(Rituxan) to form combination therapies R-CHOP, hyperCVAD-R, and R-FCM.Other approaches include combining any of the abovementioned therapieswith stem cell transplantation or treatment with ICE (iphosphamide,carboplatin and etoposide).

Another approach to treating mantle cell lymphoma includes immunotherapysuch as using monoclonal antibodies like Rituximab (Rituxan). Rituximabis also effective against other indolent B-cell cancers, includingmarginal-zone lymphoma, WM, CLL and small lymphocytic lymphoma. Acombination of Rituximab and chemotherapy agents is especiallyeffective. A modified approach is radioimmunotherapy, wherein amonoclonal antibody is combined with a radioisotope particle, such asIodine-131 tositumomab (Bexxar®) and Yttrium-90 ibritumomab tiuxetan(Zevalin®). In another example, Bexxar® is used in sequential treatmentwith CHOP. Another immunotherapy example includes using cancer vaccines,which is based upon the genetic makeup of an individual patient's tumor.A lymphoma vaccine example is GTOP-99 (MyVax).

Another approach to treating mantle cell lymphoma includes autologousstem cell transplantation coupled with high-dose chemotherapy.

Another approach to treating mantle cell lymphoma includes administeringproteasome inhibitors, such as Velcade® (bortezomib or PS-341), orantiangiogenesis agents, such as thalidomide, especially in combinationwith Rituxan. Another treatment approach is administering drugs thatlead to the degradation of Bcl-2 protein and increase cancer cellsensitivity to chemotherapy, such as oblimersen (Genasense) incombination with other chemotherapeutic agents. Another treatmentapproach includes administering mTOR inhibitors, which can lead toinhibition of cell growth and even cell death; a non-limiting example isTemsirolimus (CCI-779), and Temsirolimus in combination with Rituxan®,Velcade® or other chemotherapeutic agents.

Other recent therapies for MCL have been disclosed (Nature Reviews;Jares, P. 2007). Non-limiting examples include Flavopiridol, PD0332991,R-roscovitine (Selicilib, CYC202), Styryl sulphones, Obatoclax(GX15-070), TRAIL, Anti-TRAIL DR4 and DR5 antibodies, Temsirolimus(CCl-779), Everolimus (RAD001), BMS-345541, Curcumin, Vorinostat (SAHA),Thalidomide, lenalidomide (Revlimid®, CC-5013), and Geldanamycin(17-AAG).

Non-limiting examples of other therapeutic agents used to treatWaldenstrom's Macroglobulinemia include perifosine, bortezomib(Velcade®), rituximab, sildenafil citrate (Viagra®), CC-5103,thalidomide, epratuzumab (hLL2-anti-CD22 humanized antibody),simvastatin, enzastaurin, campath-1H, dexamethasone, DT PACE,oblimersen, antineoplaston A10, antineoplaston AS2-1, alemtuzumab, betaalethine, cyclophosphamide, doxorubicin hydrochloride, prednisone,vincristine sulfate, fludarabine, filgrastim, melphalan, recombinantinterferon alfa, carmustine, cisplatin, cyclophosphamide, cytarabine,etoposide, melphalan, dolastatin 10, indium In 111 monoclonal antibodyMN-14, yttrium Y 90 humanized epratuzumab, anti-thymocyte globulin,busulfan, cyclosporine, methotrexate, mycophenolate mofetil, therapeuticallogeneic lymphocytes, Yttrium Y 90 ibritumomab tiuxetan, sirolimus,tacrolimus, carboplatin, thiotepa, paclitaxel, aldesleukin, recombinantinterferon alfa, docetaxel, ifosfamide, mesna, recombinantinterleukin-12, recombinant interleukin-11, Bcl-2 family proteininhibitor ABT-263, denileukin diftitox, tanespimycin, everolimus,pegfilgrastim, vorinostat, alvocidib, recombinant flt3 ligand,recombinant human thrombopoietin, lymphokine-activated killer cells,amifostine trihydrate, aminocamptothecin, irinotecan hydrochloride,caspofungin acetate, clofarabine, epoetin alfa, nelarabine, pentostatin,sargramostim, vinorelbine ditartrate, WT-1 analog peptide vaccine, WT1126-134 peptide vaccine, fenretinide, ixabepilone, oxaliplatin,monoclonal antibody CD19, monoclonal antibody CD20, omega-3 fatty acids,mitoxantrone hydrochloride, octreotide acetate, tositumomab and iodine1-131 tositumomab, motexafin gadolinium, arsenic trioxide, tipifarnib,autologous human tumor-derived HSPPC-96, veltuzumab, bryostatin 1, andPEGylated liposomal doxorubicin hydrochloride, and any combinationthereof.

Non-limiting examples of other therapeutic agents used to treat diffuselarge B-cell lymphoma (DLBCL) drug therapies (Blood 2005 Abramson, J.)include cyclophosphamide, doxorubicin, vincristine, prednisone,anti-CD20 monoclonal antibodies, etoposide, bleomycin, many of theagents listed for Waldenstrom's, and any combination thereof, such asICE and R-ICE.

Non-limiting examples of therapeutic procedures used to treatWaldenstrom's Macroglobulinemia include peripheral blood stem celltransplantation, autologous hematopoietic stem cell transplantation,autologous bone marrow transplantation, antibody therapy, biologicaltherapy, enzyme inhibitor therapy, total body irradiation, infusion ofstem cells, bone marrow ablation with stem cell support, invitro-treated peripheral blood stem cell transplantation, umbilical cordblood transplantation, immunoenzyme technique, pharmacological study,low-LET cobalt-60 gamma ray therapy, bleomycin, conventional surgery,radiation therapy, and nonmyeloablative allogeneic hematopoietic stemcell transplantation.

Non-limiting examples of other therapeutic agents used to treat ChronicLymphocytic Leukemia (Spectrum, 2006, Fernandes, D.) includeChlorambucil (Leukeran), Cyclophosphamide (Cyloxan, Endoxan, Endoxana,Cyclostin), Fludarabine (Fludara), Pentstatin (Nipent), Cladribine(Leustarin), Doxorubicin (Adriamycin®, Adriblastine), Vincristine(Oncovin), Prednisone, Prednisolone, Alemtuzumab (Campath, MabCampath),many of the agents listed for Waldenstrom's, and combinationchemotherapy and chemoimmunotherapy, including the common combinationregimen: CVP (cyclophosphamide, vincristine, prednisone); R-CVP(rituximab-CVP); ICE (iphosphamide, carboplatin, etoposide); R-ICE(rituximab-ICE); FCR (fludarabine, cyclophosphamide, rituximab); and FR(fludarabine, rituximab).

In certain embodiments, the method comprises administering in additionto a compound of I or II to said patient, a therapeutically effectiveamount of at least one therapeutic agent and/or therapeutic procedureselected to treat said cancer or autoimmune disease in said patient. Incertain embodiments, the method comprises administering in addition to acompound of I or II to said patient, a therapeutically effective amountof a combination of therapeutic agents selected from the groupconsisting of a) CHOP (cyclophosphamide, doxorubicin, vincristine,prednisone); b) R-CHOP (rituximab-CHOP); c) hyperCVAD (hyperfractionatedcyclophosphamide, vincristine, doxorubicin, dexamethasone, methotrexate,cytarabine); d) R-hyperCVAD (rituximab-hyperCVAD); e) FCM (fludarabine,cyclophosphamide, mitoxantrone); f) R-FCM (rituximab, fludarabine,cyclophosphamide, mitoxantrone); g) bortezomib and rituximab; h)temsirolimus and rituximab; i) temsirolimus and Velcade®; j) Iodine-131tositumomab (Bexxar®) and CHOP; k) CVP (cyclophosphamide, vincristine,prednisone); l) R-CVP (rituximab-CVP); m) ICE (iphosphamide,carboplatin, etoposide); n) R-ICE (rituximab-ICE); o) FCR (fludarabine,cyclophosphamide, rituximab); and p) FR (fludarabine, rituximab).

The compounds of the invention may be formulated for administration toanimal subject using commonly understood formulation techniques wellknown in the art. Formulations which are suitable for particular modesof administration and for the compounds of formula A may be found inRemington's Pharmaceutical Sciences, latest edition, Mack PublishingCompany, Easton, Pa.

The compounds of the invention may be prepared in the form of prodrugs,i.e., protected forms which release the compounds of the invention afteradministration to the subject. Typically, the protecting groups arehydrolyzed in body fluids such as in the bloodstream thus releasing theactive compound or are oxidized or reduced in vivo to release the activecompound. A discussion of prodrugs is found in Smith and WilliamsIntroduction to the Principles of Drug Design, Smith, H. J.; Wright,2^(nd) ed., London (1988).

A compound of the present invention can be administered as the neatchemical, but it is typically preferable to administer the compound inthe form of a pharmaceutical composition or formulation. Accordingly,the present invention also provides pharmaceutical compositions thatcomprise a compound of formula A and a biocompatible pharmaceuticalcarrier, adjuvant, or vehicle. The composition can include the compoundof Formula A as the only active moiety or in combination with otheragents, such as oligo- or polynucleotides, oligo- or polypeptides,drugs, or hormones mixed with excipient(s) or other pharmaceuticallyacceptable carriers. Carriers and other ingredients can be deemedpharmaceutically acceptable insofar as they are compatible with otheringredients of the formulation and not deleterious to the recipientthereof.

The pharmaceutical compositions are formulated to contain suitablepharmaceutically acceptable carriers, and can optionally compriseexcipients and auxiliaries that facilitate processing of the activecompounds into preparations that can be used pharmaceutically. Theadministration modality will generally determine the nature of thecarrier. For example, formulations for parenteral administration cancomprise aqueous solutions of the active compounds in water-solubleform. Carriers suitable for parenteral administration can be selectedfrom among saline, buffered saline, dextrose, water, and otherphysiologically compatible solutions. Preferred carriers for parenteraladministration are physiologically compatible buffers such as Hank'ssolution, Ringer's solution, or physiologically buffered saline. Fortissue or cellular administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art. For preparations comprisingproteins, the formulation can include stabilizing materials, such aspolyols (e.g., sucrose) and/or surfactants (e.g., nonionic surfactants),and the like.

Alternatively, formulations for parenteral use can comprise dispersionsor suspensions of the active compounds prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, and synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Aqueous injectionsuspensions can contain substances that increase the viscosity of thesuspension, such as sodium carboxy-methylcellulose, sorbitol, ordextran. Optionally, the suspension also can contain suitablestabilizers or agents that increase the solubility of the compounds toallow for the preparation of highly concentrated solutions. Aqueouspolymers that provide pH-sensitive solubilization and/or sustainedrelease of the active agent also can be used as coatings or matrixstructures, e.g., methacrylic polymers, such as the Eudragit® seriesavailable from Rohm America Inc. (Piscataway, N.J.). Emulsions, e.g.,oil-in-water and water-in-oil dispersions, also can be used, optionallystabilized by an emulsifying agent or dispersant (surface activematerials; surfactants). Suspensions can contain suspending agents suchas ethoxylated isostearyl alcohols, polyoxyethlyene sorbitol andsorbitan esters, microcrystalline cellulose, aluminum metahydroxide,bentonite, agar-agar, gum tragacanth, and mixtures thereof.

Liposomes containing the active compound of Formula A also can beemployed for parenteral administration. Liposomes generally are derivedfrom phospholipids or other lipid substances. The compositions inliposome form also can contain other ingredients, such as stabilizers,preservatives, excipients, and the like. Preferred lipids includephospholipids and phosphatidyl cholines (lecithins), both natural andsynthetic. Methods of forming liposomes are known in the art. See,.e.g., Prescott (Ed.), Methods in Cell Biology, Vol. XIV, p. 33, AcademicPress, New York (1976).

The pharmaceutical compositions comprising the compound of Formula A indosages suitable for oral administration can be formulated usingpharmaceutically acceptable carriers well known in the art. Thepreparations formulated for oral administration can be in the form oftablets, pills, capsules, cachets, dragees, lozenges, liquids, gels,syrups, slurries, elixirs, suspensions, or powders. To illustrate,pharmaceutical preparations for oral use can be obtained by combiningthe active compounds with a solid excipient, optionally grinding theresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries if desired, to obtain tablets or dragée cores. Oralformulations can employ liquid carriers similar in type to thosedescribed for parenteral use, e.g., buffered aqueous solutions,suspensions, and the like.

Preferred oral formulations include tablets, dragees, and gelatincapsules. These preparations can contain one or excipients, whichinclude, without limitation:

a) diluents, such as sugars, including lactose, dextrose, sucrose,mannitol, or sorbitol;

b) binders, such as magnesium aluminum silicate, starch from corn,wheat, rice, potato, etc.;

c) cellulose materials, such as methylcellulose, hydroxypropylmethylcellulose, and sodium carboxymethylcellulose, polyvinylpyrrolidone,gums, such as gum arabic and gum tragacanth, and proteins, such asgelatin and collagen;

d) disintegrating or solubilizing agents such as cross-linked polyvinylpyrrolidone, starches, agar, alginic acid or a salt thereof, such assodium alginate, or effervescent compositions;

e) lubricants, such as silica, talc, stearic acid or its magnesium orcalcium salt, and polyethylene glycol;

f) flavorants and sweeteners;

g) colorants or pigments, e.g., to identify the product or tocharacterize the quantity (dosage) of active compound; and

h) other ingredients, such as preservatives, stabilizers, swellingagents, emulsifying agents, solution promoters, salts for regulatingosmotic pressure, and buffers.

In some preferred oral formulations, the pharmaceutical compositioncomprises at least one of the materials from group (a) above, or atleast one material from group (b) above, or at least one material fromgroup (c) above, or at least one material from group (d) above, or atleast one material from group (e) above. Preferably, the compositioncomprises at least one material from each of two groups selected fromgroups (a)-(e) above.

Gelatin capsules include push-fit capsules made of gelatin, as well assoft, sealed capsules made of gelatin and a coating such as glycerol orsorbitol. Push-fit capsules can contain the active ingredient(s) mixedwith fillers, binders, lubricants, and/or stabilizers, etc. In softcapsules, the active compounds can be dissolved or suspended in suitablefluids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycol with or without stabilizers.

Dragée cores can be provided with suitable coatings such as concentratedsugar solutions, which also can contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide,lacquer solutions, and suitable organic solvents or solvent mixtures.

The pharmaceutical composition can be provided as a salt of the activecompound. Salts tend to be more soluble in aqueous or other protonicsolvents than the corresponding free acid or base forms.Pharmaceutically acceptable salts are well known in the art. Compoundsthat contain acidic moieties can form pharmaceutically acceptable saltswith suitable cations. Suitable pharmaceutically acceptable cationsinclude, for example, alkali metal (e.g., sodium or potassium) andalkaline earth (e.g., calcium or magnesium) cations.

Compounds of structural formula (A) that contain basic moieties can formpharmaceutically acceptable acid addition salts with suitable acids. Forexample, Berge, et al., describe pharmaceutically acceptable salts indetail in J Pharm Sci, 66:1 (1977). The salts can be prepared in situduring the final isolation and purification of the compounds of theinvention or separately by reacting a free base function with a suitableacid.

Representative acid addition salts include, but are not limited to,acetate, adipate, alginate, citrate, aspartate, benzoate,benzenesulfonate, bisulfate, butyrate, camphorate, camphorolsulfonate,digluconate, glycerophosphate, hemisulfate, heptanoate, hexanoate,fumarate, hydrochloride, hydrobromide, hydroiodide,2-hydroxyethanesulfonate (isothionate), lactate, maleate,methanesulfonate or sulfate, nicotinate, 2-naphthalenesulfonate,oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate,pivalate, propionate, succinate, tartrate, thiocyanate, phosphate orhydrogen phosphate, glutamate, bicarbonate, p-toluenesulfonate, andundecanoate. Examples of acids that can be employed to formpharmaceutically acceptable acid addition salts include, withoutlimitation, such inorganic acids as hydrochloric acid, hydrobromic acid,sulfuric acid, and phosphoric acid, and such organic acids as oxalicacid, maleic acid, succinic acid, and citric acid.

Basic nitrogen-containing groups can be quaternized with such agents aslower alkyl halides such as methyl, ethyl, propyl, and butyl chlorides,bromides and iodides; dialkyl sulfates like dimethyl, diethyl, dibutyl,and diamyl sulfates; long chain alkyl halides such as decyl, lauryl,myristyl, and stearyl chlorides, bromides, and iodides; arylalkylhalides such as benzyl and phenethyl bromides; and others. Productshaving modified solubility or dispersibility are thereby obtained.

Compositions comprising a compound of the invention formulated in apharmaceutical acceptable carrier can be prepared, placed in anappropriate container, and labeled for treatment of an indicatedcondition. Accordingly, there also is contemplated an article ofmanufacture, such as a container comprising a dosage form of a compoundof the invention and a label containing instructions for use of thecompound. Kits are also contemplated under the invention. For example,the kit can comprise a dosage form of a pharmaceutical composition and apackage insert containing instructions for use of the composition intreatment of a medical condition. In either case, conditions indicatedon the label can include treatment of inflammatory disorders, cancer,etc.

Methods of Administration

Pharmaceutical compositions comprising a compound of formula A can beadministered to the subject by any conventional method, includingparenteral and enteral techniques. Parenteral administration modalitiesinclude those in which the composition is administered by a route otherthan through the gastrointestinal tract, for example, intravenous,intraarterial, intraperitoneal, intramedullarly, intramuscular,intraarticular, intrathecal, and intraventricular injections. Enteraladministration modalities include, for example, oral (including buccaland sublingual) and rectal administration. Transepithelialadministration modalities include, for example, transmucosaladministration and transdermal administration. Transmucosaladministration includes, for example, enteral administration as well asnasal, inhalation, and deep lung administration; vaginal administration;and rectal administration. Transdermal administration includes passiveor active transdermal or transcutaneous modalities, including, forexample, patches and iontophoresis devices, as well as topicalapplication of pastes, salves, or ointments. Parenteral administrationalso can be accomplished using a high-pressure technique, e.g.,POWDERJECT™.

Surgical techniques include implantation of depot (reservoir)compositions, osmotic pumps, and the like. A preferred route ofadministration for treatment of inflammation can be local or topicaldelivery for localized disorders such as arthritis, or systemic deliveryfor distributed disorders, e.g., intravenous delivery for reperfusioninjury or for systemic conditions such as septicemia. For otherdiseases, including those involving the respiratory tract, e.g., chronicobstructive pulmonary disease, asthma, and emphysema, administration canbe accomplished by inhalation or deep lung administration of sprays,aerosols, powders, and the like.

In some foregoing embodiments, the compound of formula A is administeredbefore, during, or after administration of chemotherapy, radiotherapy,and/or surgery. The formulation and route of administration chosen willbe tailored to the individual subject, the nature of the condition to betreated in the subject, and generally, the judgment of the attendingpractitioner.

The therapeutic index of the compound of formula A can be enhanced bymodifying or derivatizing the compounds for targeted delivery to cancercells expressing a marker that identifies the cells as such. Forexample, the compounds can be linked to an antibody that recognizes amarker that is selective or specific for cancer cells, so that thecompounds are brought into the vicinity of the cells to exert theireffects locally, as previously described (see for example, Pietersz, etal., Immunol Rev, 129:57 (1992); Trail, et al., Science, 261:212 (1993);and Rowlinson-Busza, et al., Curr Opin Oncol, 4:1142 (1992)).Tumor-directed delivery of these compounds enhances the therapeuticbenefit by, inter alia, minimizing potential nonspecific toxicities thatcan result from radiation treatment or chemotherapy. In another aspect,the compound of formula A and radioisotopes or chemotherapeutic agentscan be conjugated to the same anti-tumor antibody.

The characteristics of the agent itself and the formulation of the agentcan influence the physical state, stability, rate of in vivo release,and rate of in vivo clearance of the administered agent. Suchpharmacokinetic and pharmacodynamic information can be collected throughpreclinical in vitro and in vivo studies, later confirmed in humansduring the course of clinical trials. Thus, for any compound used in themethod of the invention, a therapeutically effective dose can beestimated initially from biochemical and/or cell-based assays. Then,dosage can be formulated in animal models to achieve a desirablecirculating concentration range that modulates expression or activity ofa particular PI3K isoform or combination of isoforms. As human studiesare conducted, further information will emerge regarding the appropriatedosage levels and duration of treatment for various diseases andconditions.

Although compounds of the invention are well tolerated, an example of alimit to the treatment dosage is elevated liver function tests (LFT).LFT involve standard clinical biochemistry tests on the patient's serumor plasma to provide information about the state of a patient's liver.Levels, such as alanine transaminase, aspartate transaminase, alkalinephosphatase, bilirubin, and gamma glutamyl transpeptidase, that areoutside the normal range can signal possible liver toxicity. Dosing ofthe therapeutic compound can be adjusted to avoid or reduce elevatedliver function test values and subsequent potential for liver toxicity.For instance, a subject may be administered escalating doses of acompound. At a certain dose amount, the subject begins to developelevated LFT levels outside a normal range, signaling potential livertoxicity at that dosage. In response, the dosage may be reduced to anamount such that LFT levels are reduced to an acceptable range as judgedby the treating physician, e.g. a level that is in the range normal forthe subject being treated, or within about 25% to 50% of normal.Therefore, liver function tests can be used to titrate theadministration dosage of a compound.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe “therapeutic index,” which typically is expressed as the ratioLD50/ED50. Compounds that exhibit large therapeutic indices, i.e., thetoxic dose is substantially higher than the effective dose, arepreferred. The data obtained from such cell culture assays andadditional animal studies can be used in formulating a range of dosagefor human use. The dosage of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity.

Dosage may be limited by treatment-related toxicity symptoms. Suchsymptoms besides elevated liver function tests include anemia, visionblurring, diarrhea, vomiting, fatigue, mucositis, peripheral edema,pyrexia, peripheral neuropathy, pleural effusion, night sweats, andorthopnea, or a combination thereof. At a certain dose amount, if thesubject develops intolerable levels of such symptoms, the dosage may bereduced such that the adverse event is eliminated and no longer adverseor reduced to an acceptable level as judged by a treating physician.

Another consideration in determining the appropriate dose of compoundfor a patient is the desired concentration circulating in the bloodplasma. In a particular embodiment, the concentration of compound in theblood is between 40-3,000 ng/mL over a 12 hour period from the time ofadministration. In another particular embodiment, the concentration ofcompound in the blood is between 75-2,000 ng/mL over a 12 hour periodfrom the time of administration. In another particular embodiment, theconcentration of compound in the blood is between 500-2,000 ng/mL over a12 hour period from the time of administration. In a preferredembodiment, the concentration of compound in the blood is between40-3,000 ng/mL over a 12 hour period from the time of administration,wherein the compound is a formula of I, I″, II, or II″ and is orallyadministered in an amount of about 50 mg, 100 mg, 150 mg, or 200 mg. Ina preferred embodiment, the concentration of compound in the blood isbetween 40-3,000 ng/mL over a 12 hour period from the time ofadministration, wherein the compound is a formula of I and is orallyadministered in an amount of about 50 mg, 100 mg, 150 mg, or 200 mg. Ina preferred embodiment, the concentration of compound in the blood isbetween 40-3,000 ng/mL over a 12 hour period from the time ofadministration, wherein the compound is a formula of II and is orallyadministered in an amount of about 50 mg, 100 mg, 150 mg, or 200 mg. Insome of the foregoing embodiments, the maximum concentration in theblood plasma is achieved within two hours of administration.

In certain embodiments, the dosage of the compound of Formula I or II isselected to produce a plasma concentration of drug of about 10 nM orhigher over a period of 8 to 12 hours, on average, and to provide a peakplasma concentration of about 500 nM or higher, preferably about 1000 nMor higher. In certain embodiments, the dosage of the compound of FormulaI or II is selected to produce a plasma concentration of drug of about100 nM or higher over a period of 8 to 12 hours, on average, and toprovide a peak plasma concentration of about 500 nM or higher,preferably about 1000 nM or higher. In certain embodiments, the dosageof the compound of Formula I or II is selected to produce a plasmaconcentration of drug of about 200 nM or higher over a period of 8 to 12hours, on average, and to provide a peak plasma concentration of about500 nM or higher, preferably about 1000 nM or higher.

In certain embodiments, the dosage of the compound of formula I or II isselected to produce a plasma concentration wherein the troughconcentration of the compound is in the range where a therapeuticeffect, such as apoptosis of cancer cells, is observed. In certainembodiments, the dosage of the compound of formula I or II is selectedto produce a trough plasma concentration at or higher than the EC₅₀PI3Kδ isoform activation in blood plasma. In certain embodiments, thedosage of the compound of formula I or II is selected to produce antrough blood concentration above the EC₅₀ level for PI3Kδ activation andbelow the level for EC₅₀ PI3Kγ activation in a cell during a period ofat least 12 hours from compound administration. For instance, if theEC₅₀ value for PI3K δ basophil activation is 65 nM and the EC₅₀ valuefor PI3K γ basophil activation is 1100 nM in whole blood plasma, thenthe dosage of the compound selected provides a trough plasmaconcentration of the compound between 60 nM and 1100 nM during a periodof 8-12 hours from compound administration. Similarly, a dosage can beselected to produce an trough blood concentration above the EC₅₀ levelfor PI3Kδ basophil activation and below the EC₅₀ level for PI3K-α, -β or-γ basophil activation. The EC50 values for the PI3K isoform activationor inhibition in vivo can be determined by a person having ordinaryskill in the art. In alternative embodiments, the upper range of thetrough concentration of the drug may exceed and is not limited by theEC₅₀ value of the PI3K-γ, -α, or β isoform in blood plasma. Moreover,the blood concentration range of the drug is at a level which istherapeutically beneficial in treating the hematologic malignancy, whileminimizing undesirable side effects.

For instance, while being delta-selective, the compounds can exhibitsufficient activity on p110γ to be clinically useful, i.e., to beeffective on a cancer that relies upon p110γ for signaling, because aplasma level above the effective dosage for inhibition of p110γ can beachieved while still being selective relative to other isoforms,particularly the alpha isoform. Thus, in some embodiments, the dosage ofthe compound is selected to produce a blood concentration effective forselectively inhibiting p110δ and p110γ.

In some embodiments, the dosage of the compound provides a trough bloodplasma concentration between 65 nM and 1100 nM during a period of 8 to12 hours from compound administration. In some foregoing embodiments,the period is at least 12 hours from compound administration.

In a particular embodiment, the compound is administered in atherapeutically effective amount.

In a particular embodiment, the compound is administered at a dose of20-500 mg/day. In a particular embodiment, the compound is administeredat a dose of 50-250 mg/day.

In a particular embodiment, the compound is administered at a dose of 25to 150 mg per dose, and two doses are administered per day (e.g., BIDdosing with 25 to 150 mg doses). In a preferred embodiment, a subject istreated with 50 mg to 100 mg of a compound of formula A twice per day.In another preferred embodiment, a subject is treated with 50 mg to 150mg of a compound of formula A twice per day.

In a particular embodiment, the method comprises administering to saidpatient an initial daily dose of 20-500 mg of the compound andincreasing said dose by increments until clinical efficacy is achieved.Increments of about 25, 50, or 100 mg can be used to increase the dose.The dosage can be increased daily, every other day, twice per week, oronce per week.

In a particular embodiment, the method comprises continuing to treatsaid patient by administering the same dose of the compound at whichclinical efficacy is achieved or reducing said dose by increments to alevel at which efficacy can be maintained.

In a particular embodiment, the method comprises administering to saidpatient an initial daily dose of 20-500 mg of the compound andincreasing said dose to a total dosage of 50-400 mg per day over atleast 6 days. Optionally, the dosage can be further increased to about750 mg/day.

In a particular embodiment, the compound is administered at least twicedaily.

In a particular embodiment, the compound is administered orally,intravenously or by inhalation. Preferably, the compound is administeredorally. In some embodiments, it is administered orally at a dosage ofabout 50 mg BID or at a dosage of about 100 mg BID. In otherembodiments, it is administered orally at a dosage of about 150 mg BID.

For the methods of the invention, any effective administration regimenregulating the timing and sequence of doses can be used. Doses of theagent preferably include pharmaceutical dosage units comprising aneffective amount of the agent. As used herein, “effective amount” refersto an amount sufficient to modulate PI3Kδ expression or activity and/orderive a measurable change in a physiological parameter of the subjectthrough administration of one or more of the pharmaceutical dosageunits. “Effective amount” can also refer to the amount required toameliorate a disease or disorder in a subject.

Suitable dosage ranges for the compounds of formula A vary according tothese considerations, but in general, the compounds are administered inthe range of 10.0 μg/kg-15 mg/kg of body weight; 1.0 μg/kg-10 mg/kg ofbody weight, or 0.5 mg/kg-5 mg/kg of body weight. For a typical 70-kghuman subject, thus, the dosage range is from 700 μg-1050 mg; 70 μg-700mg; or 35 mg-350 mg per dose, and two or more doses may be administeredper day. Dosages may be higher when the compounds are administeredorally or transdermally as compared to, for example, i.v.administration. The reduced toxicity of a compound of formula A, permitsthe therapeutic administration of relatively high doses. In some of theforegoing embodiments, oral administration of up to 750 mg/day of acompound of the invention is suitable. In some of the foregoingembodiments, a compound of formula A is administered at a dose of 50 mgBID. In some of the foregoing embodiments, a compound of formula A isadministered at a dose of 100 mg BID. In some of the foregoingembodiments, a compound of formula A is administered at a dosage of 150mg BID. In some of the foregoing embodiments, a compound of formula A isadministered at a dose of 200 mg BID. In some of the foregoingembodiments, a compound of formula A is administered at a dose of 350 mgBID. In specific embodiments, for treatment of leukemias, lymphomas andmultiple myeloma, a dosage of about 50-350 mg per dose, administeredorally once or preferably twice per day, is often suitable.

In some of the foregoing embodiments, oral administration of up to 750mg/day of compound I″ or II″ is suitable. In some of the foregoingembodiments, a compound of formula I″ or II″ is administered at a doseof 50 mg BID. In some of the foregoing embodiments, a compound offormula I″ or II″ is administered at a dose of 100 mg BID. In some ofthe foregoing embodiments, a compound of formula I″ or II″ isadministered at a dose of 150 mg BID. In some of the foregoingembodiments, a compound of formula I″ or II″ is administered at a doseof 200 mg BID. I In some of the foregoing embodiments, a compound offormula I″ or II″ is administered at a dose of 350 mg BID. In some ofthe foregoing embodiments, for treatment of leukemias, lymphomas andmultiple myeloma, a dosage of about 50-350 mg per dose of a compound offormula I″ or II″, administered orally once or preferably twice per day,is often suitable.

The compounds may be administered as a single bolus dose, a dose overtime, as in i.v. or transdermal administration, or in multiple dosages.

Dosing may be continued for at least seven days. In some embodiments,daily dosing is continued for about 28 days. In some embodiments, dosingis continued for about 28 days and is then discontinued for at least 7days. In some embodiments, a complete cycle is continuous daily dosingfor 28 days. Evaluation of a clinical response in the patient can bemeasured after each cycle. The clinical results can be used to make adecision to increase, decrease, discontinue or maintain the dosage.

Depending on the route of administration, a suitable dose can becalculated according to body weight, body surface area, or organ size.The final dosage regimen will be determined by the attending physicianin view of good medical practice, considering various factors thatmodify the action of drugs, e.g., the agent's specific activity, theidentity and severity of the disease state, the responsiveness of thepatient, the age, condition, body weight, sex, and diet of the patient,and the severity of any infection. Additional factors that can be takeninto account include time and frequency of administration, drugcombinations, reaction sensitivities, and tolerance/response to therapy.Further refinement of the dosage appropriate for treatment involving anyof the formulations mentioned herein is done routinely by the skilledpractitioner without undue experimentation, especially in light of thedosage information and assays disclosed, as well as the pharmacokineticdata observed in human clinical trials. Appropriate dosages can beascertained through use of established assays for determiningconcentration of the agent in a body fluid or other sample together withdose response data.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe compound of Formula A and the route of administration. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Accordingly, thepharmaceutical compositions can be administered in a single dose,multiple discrete doses, continuous infusion, sustained release depots,or combinations thereof, as required to maintain desired minimum levelof the compound. Short-acting pharmaceutical compositions (i.e., shorthalf-life) can be administered once a day or more than once a day (e.g.,two, three, or four times a day). Long acting pharmaceuticalcompositions might be administered every 3 to 4 days, every week, oronce every two weeks. Pumps, such as subcutaneous, intraperitoneal, orsubdural pumps, can be preferred for continuous infusion.

Subjects that will respond favorably to the method of the inventioninclude medical and veterinary subjects generally, including humanpatients. Among other subjects for whom the methods of the invention isuseful are cats, dogs, large animals, avians such as chickens, and thelike. In general, any subject who would benefit from a compound offormula A is appropriate for administration of the invention method. Insome foregoing embodiments, the patient has a cytogenetic characteristicof del(17p) or del(11q). In some foregoing, embodiments, the patient hasa lymphadenopathy. In some foregoing embodiments, the use of compound I,I″, II, or II″ reduces the size of a lymphadenopathy in a patient. Insome foregoing embodiments, the use of compound I, I″, II, or II″reduces the size of a lymphadenopathy after one cycle of treatment. Insome foregoing embodiments, the use of compound I, I″, II, or II″reduces the size of a lymphadenopathy by at least 10% after one cycle oftreatment. In some foregoing embodiments, the use of compound I, I″, II,or II″ reduces the size of a lymphadenopathy by at least 25% after onecycle of treatment. In some foregoing embodiments, the use of compoundI, I″, II, or II″ reduces the size of a lymphadenopathy by at least 30%after one cycle of treatment. In some foregoing embodiments, the use ofcompound I, I″, II, or II″ reduces the size of a lymphadenopathy by atleast 40% after one cycle of treatment. In some foregoing embodiments,the use of compound I, I″, II, or II″ reduces the size of alymphadenopathy by at least 50% after one cycle of treatment. In someforegoing embodiments, the use of compound I, I″, II, or II″ reduces thesize of a lymphadenopathy by at least 75% after one cycle of treatment.

In one aspect, the invention provides a method of treating a condition,comprising administering a compound of formula I, II or apharmaceutically acceptable salt thereof and one or more therapeuticagents to a subject in need of such treatment, wherein the condition isa cancer or an autoimmune condition. In preferred embodiments, thetherapeutic agent is a proteasome inhibitor. In more specificembodiments, the therapeutic agent is bortezomib. In some of theforegoing embodiments, the condition is a hematologic malignancy. Inpreferred embodiments, the condition is selected from the groupconsisting of multiple myeloma, acute lymphocytic leukemia, acutemyeloid leukemia, chronic lymphocytic leukemia, B-cell lymphoma, diffuselarge B-cell lymphoma, B-cell ALL, T-cell ALL and Hodgkin's lymphoma. Inpreferred embodiments, the compound is substantially comprised of theS-enantiomer. In specific embodiments, the compound comprises at least95% of the S-enantiomer. In some of the foregoing embodiments, theadministration of said compound and therapeutic agent provides asynergistic benefit superior to results obtained without the combinationof the compound and therapeutic agent.

The following examples are offered to illustrate but not to limit theinvention. In the examples below, references to the ‘compound of formulaI’ or ‘compound I’ refer to the S-enantiomer shown here, and samplesused for these Examples exhibited a 98.2% ee as measured by chiral HPLCmethods:

In addition, an analysis of this compound reveals the followingcharacteristics of the material:

Test Test Result Appearance Slightly off-white powder 1H-NMR Spectrumconforms to the reference HPLC Assay 98.1% (Anhydrous, solvent-freebasis) Chiral Purity 98.2% ee (HPLC)

Test Test Result Residual on Ignition 0.11% Infrared SpectroscopySpectrum in agreement (FTIR) with the reference 13C-NMR Spectrumconforms to the reference Particle Size Analysis Median diameter: 11.3μm Water (Coulometric 0.56% Karl Fischer)

Test Result Property or Test Expected Found Elemental Analysis % C 63.363.5 % C, H, F, N % H 4.4 4.4 % N 23.5 23.1 % F 4.5 4.5

Example 1 Inhibition of Cell Growth in MM Cells

This example demonstrates the compound of formula I inhibits thecellular growth stimulatory effects of cytokines (IGF-1 and IL-6) inmultiple myeloma (MM) cells. LB cells (Myelomonocytic myeloma cell line)were cultured for 48 h with control media; with the compound of formulaI, in the presence or absence of either IL-6 or IGF-1. The inhibitoryeffect of the compound of formula I on MM cell growth was assessed bymeasuring 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrasodium bromide(MTT; Chemicon International) dye absorbance. Cells were pulsed with 10μL of 5 mg/mL MTT to each well for the last 4 hours of 48-hour cultures,followed by 100 μL isopropanol containing 0.04 N HCl. Absorbance wasmeasured at 570/630 nm using a spectrophotometer (Molecular Devices). Asummary of the results is shown in FIG. 1. Exposure of 0.625 μM-2.5 μMof Compound I inhibits MM cell growth even in the presence of cellgrowth stimulatory cytokines.

Example 2 Effect of BMSC on Cytotoxicity

This example demonstrates Bone Marrow Stromal Cells (BMSCs) do notprotect against compound I-induced LB cell cytotoxicity. LB cells werecultured with control media, and with the compound of formula I for 48hours, in the presence or absence of BMSCs. Cell proliferation wasassessed using [³H]-thymidine uptake assay. All data represent mean(±SD) of triplicate experiment. A summary of the results is shown inFIG. 2. LB cell growth is reduced after exposure to 0.625 μM-10 μM ofcompound I even in the presence of BMSC.

Example 3 Effect of Compound on Apoptosis of CLL Cells

This example demonstrates the compound of formula I induces apoptosis inpatient chronic lymphocytic leukemia (CLL) cells. Peripheral blood wasobtained from patients with B-CLL through the CLL Research Consortiumfrom Ohio State University. Primary CD19-positive cells were isolatedusing Rosette-Sep (StemCell Technologies). Cells were maintained in RPMI1640 (Invitrogen) supplemented with 10% heat-inactivated fetal bovineserum, 2 mmol/L L-glutamine, and penicillin (100 units/mL)/streptomycin(100 μg/mL; Invitrogen) at 37° C., 5% CO₂, and high humidity. Afterincubation with the compound of formula I or medium for 96 hours, 5×10⁵cells were washed with PBS and then resuspended in binding buffer (10mmol/L HEPES/NaOH, pH 7.4, 150 mmol/L NaCl 5 mmol/L KCl, 1 mmol/L MgCl₂,1.8 mmol/L CaCl₂) containing 2 μL of Annexin V-FITC stock (BioWhittaker,Inc) and 10 μL of 20 μg/mL PI (Sigma). After incubation for 10 minutesat room temperature in a light-protected area, the specimens werequantified by flow cytometry on a FACScan™ (Becton Dickinson).

Treatment of CLL patient cells with compound I results in apoptosis andthe result appears to be dose-dependent, as seen in FIG. 3.

Compound I induced apoptosis was seen in CLL cells from poor prognosispatients, as the data indicates in FIG. 19.

Compound I induced apoptosis was also seen to be effective in CLL cellsfrom refractory/relapsed patients as shown in FIG. 20.

Example 4 Effect of Compound in ALL Cell Lines

This example demonstrates the compound of formula I results in areduction of Akt phosphorylation and a decrease in cellularproliferation accompanied by cell death in both T-ALL and B-ALL (AcuteLymphoblastic Leukemia) leukemic cell lines. Viability assays of celllines were performed using the AlamarBlue assay (Invitrogen). Cells(1×10⁶ per well) in a volume of 100 μL were placed in a 96-wellflat-bottom plate and the compound of formula I (100 μL per well at 2×final concentration) or medium alone was added to the plates. All wereperformed in quadruplicate. Cells were incubated for fixed times (48hours). After the incubation, 10 μL AlamarBlue® was added to each well.Cells were incubated for 4 hours and the optical density at 530-560 nmwas obtained using a SpectraMax® M5 plate reader 2001. Cell viabilitywas expressed as a percentage of absorption between treatedcells/control sample. These results are summarized in the table shown inFIG. 4. Exposure to compound I result in substantial reduction incellular viability in a variety ALL cell lines as well as reduction inAkt phosphorylation.

Example 5 Effect of Compound on ALL Cell Cycle

This example demonstrates treatment of the acute lymphoblastic leukemia(ALL) cell line CCRF-SB with the compound of formula I results in G0/G1cell cycle arrest. Representative fluorescence-activated cell sorting(FACS) analysis of propidium iodide-stained CCRF-SB cells under normalgrowth conditions, and growth in the presence of the compound of formulaI. The average percentage of cells in G₀-G₁, S, and G₂-M phases iscalculated in the table below the histographs. Results are shown in FIG.5.

Example 6 Inhibition of Proliferation of Breast Cancer Cells

This example demonstrates the compound of formula I inhibitsproliferation of breast cancer cell lines. T47D and HS-578T cell lineswere grown in the presence of serum plus the indicated concentrations ofthe compound of formula I. Proliferation was measured in triplicatewells by AlamarBlue® assay (Invitrogen) 96-well plates. Results ofproliferation assays are expressed as the mean cellular percentagevalues and shown in FIG. 6.

Example 7 Inhibition of Proliferation of Ovarian Cancer Cell Lines

This example demonstrates the compound of formula I inhibitsproliferation of ovarian cancer cell lines. IGROV-1 and OVCAR-3 celllines were grown in the presence of serum plus the indicatedconcentrations of the compound of formula I. Proliferation was measuredin triplicate wells by AlamarBlue assay (Invitrogen) 96-well plates.Results of proliferation assays are expressed as the mean cellularpercentage values and are shown in FIG. 7.

Example 8 Reduction of Akt Phosphorylation

This example demonstrates the compound of formula I reduces constitutiveAkt phosphorylation in hematopoietic tumor cell lines that exhibitedconstitutive Akt phosphorylation. A large panel of leukemia and lymphomacell lines was assessed for constitutive Akt phosphorylation. These celllines represent B-lymphoma, T-lymphoma, ALL, Malignant histiocytosis,DLBCL and AML. Cell lines that demonstrated serum independent Aktphosphorylation were treated with the compound of formula I for 2 hours.Thereafter, cell were lysed, size-fractioned and immunoblotted withantibodies directed against phospho-Akt(Ser473). Results are shown inFIG. 8. Reduction in Akt(Ser473) was achieved for all cell lines afterexposure to compound I.

Example 9 Compound I Effective in DLBCL

This example provides evidence that compound I blocks PI3K signaling andinduces apoptosis in diffuse large B-cell lymphoma cells. P110δ isexpressed in DLBCL cell lines as shown in FIG. 26A. FIG. 26B shows thatexposure to compound I reduces pAKT levels in several DLBCL cell lines.

Example 10 Inducement of Apoptosis in Breast Cancer Cells

This example demonstrates the compound of formula I induces apoptosis inbreast cancer cell lines. HS-578T, T47D, and MCF7 cells were treatedwith the compound of formula I or corresponding DMSO concentrations for24 h. The percentage of apoptotic cells was determined by AnnexinV-FITC/7AAD staining Bottom left, viable cells (Annexin V-FITC/PInegative); bottom right, early apoptotic cells (Annexin V-FITC positiveonly); top right, mid-late apoptotic cells (Annexin V-FITC/7AADdouble-positive); and top left, late apoptotic/necrotic (7AAD positiveonly). Percentages of cells in each quadrant are indicated except forthe bottom left quadrant (viable cells). One experiment representativeof three different experiments that gave similar results is shown inFIG. 10.

Example 11 Steady State Blood Levels on Day 7 in Healthy Volunteers

This example provides data relating to the concentration of the compoundof formula I in the blood of a healthy human subject on day 7. Theconcentration was monitored over a period of 12 hours, after oraladministration of 50, 100, or 200 mg BID of the compound of formula I onday 7 of the study. FIG. 11 follows the plasma concentration of the drugover a period of 12 hours from administration. The maximum concentrationof drug is achieved within two hours for all doses. Administration of50, 100 or 200 mg BID of said compound results in a concentration levelthat exceeds the PI3Kδ EC₅₀ concentration in basophil for at least 12hours.

In addition, single dose studies wherein 17-400 mg of the compound offormula I was administered in healthy volunteers was carried out.Concentration of the compound in the blood was measured over 24 hoursfrom administration and results are shown in FIG. 24A. At about 6 hours,the concentration of compound I in the blood for all administered dosesis at least about 100 nM. At about 12 hours, the concentration ofcompound I in the blood for doses 50 mg and higher is over 50 nM. Themaximum concentration of compound I in the blood is achieved within 2hours of administration.

In another experiment, the mean compound I concentration was measured onthe 7th day of 50 mg BID dosing in healthy volunteers (N=6). The meantrough concentration was higher than the EC50 for PI3Kδ and the meanpeak concentration was lower than the EC50 for PI3Kγ as determined inthe whole blood basophil activation assay, FIG. 24B. This exampledemonstrates the concentration range of compound I administered at 50 mgBID is at a level that is above the ED₅₀ PI3Kδ basophil activation levelbut lower than the minimum ED₅₀ PI3Kγ basophil level activation level inwhole blood for at least 12 hours.

Table 1, below, provides an overview of the subjects in the study,wherein either a singe dose (SD) or multiple dose (MD) of the compoundof formula I is administered to a subject at varying amounts. The “n”values refer to the number of subjects in each group.

TABLE 1 Cohort Regimen Compound I Placebo 1 (n = 8) SD  17 mg (n = 6)Placebo (n = 2) 2 (n = 8) SD  50 mg (n = 6) Placebo (n = 2) 3 (n = 8) SD125 mg (n = 6) Placebo (n = 2) 4 (n = 8) SD 250 mg (n = 6) Placebo (n =2) 5 (n = 8) SD 400 mg (n = 6) Placebo (n = 2) 6 (n = 8) MD  50 mg BID ×7 d (n = 6) Placebo BID × 7 d (n = 2) 7 (n = 8) MD 100 mg BID × 7 d (n =6) Placebo BID × 7 d (n = 2) 8 (n = 8) MD 200 mg BID × 7 d (n = 6)Placebo BID × 7 d (n = 2)

Example 12 Effect on Lesions in a Patient with Mantle Cell Lymphoma

This example provides data relating to the area of lesions of a patientwith mantle cell lymphoma after 1 cycle of treatment (28 days) with thecompound of formula I. The area of 6 lesions was measured prior totreatment and after a cycle of treatment. The response to 28 days oforal administration of 50 mg BID of the compound of formula I, resultsin a decrease of lesion area compared to area prior to treatment andrepresents a 44% decrease in tumor burden. The results are summarized ina bar graph found in FIG. 12.

Example 13 Response of a Patient with CLL to Treatment

This example provides data relating to the concentration of absolutelymphocyte count (ALC) in the blood of a patient with CLL after 1 cycle(28 days) of treatment with oral administration of the compound offormula I. The blood ALC concentration was measured over a period of 4weeks after completion of one cycle of treatment. A 55% decrease inlymphocytosis and a 38% decrease in lymphadenopathy as a result oftreatment were observed. A marked decrease in ALC concentration isobserved between week 1 and week 2, FIG. 13.

Example 14 Comparison of Lymphoma Patient to Healthy Volunteer

This example provides data comparing the concentration of the compoundof formula I in a lymphoma patient to normal healthy volunteers. On the28th day of oral administration of 50 mg BID of compound in a patientwith mantle cell lymphoma, the concentration of the compound in theblood was measured over a period of 6 hours after administration. Theconcentration of 50 and 100 mg oral administration in normal healthyvolunteers on day 7 of administration was also observed. The results aresummarized in FIG. 14. Thus, the compound does not build up excessivelyover the course of a cycle of treatment, nor does the patient becometolerant by increased metabolism over the course of the treatment cycle.

Example 15 Activity of Compound I in Various Kinases

This example shows the IC₅₀ profile of compound I across classes ofkinases as summarized in Table 2. While especially active on p110δ,Compound I was also active on p110γ and even active enough to betherapeutically useful at non-toxic doses against p110β, due to thedemonstrated high NOAEL level of the compound; while exhibiting littleactivity on Class II-V phosphoinositide kinases. Thus while beingdelta-selective, the compounds can exhibit sufficient activity on p110γto be clinically useful, i.e., to be effective on a cancer that reliesupon p110γ for signaling, because a plasma level above the effectivedosage for inhibition of p110γ can be achieved while still beingselective relative to other isoforms, particularly the alpha isoform.

TABLE 2 Other Class I PI3Ks, Class II PI3K, Class III PI3K, Class IVPI3K, Phosphoinositide IC₅₀ (nM) IC₅₀(nM) IC₅₀(nM) IC₅₀ (nM) kinasesCompound P110α p110β p110δ p110γ CIIbeta hVPS34 DNA-PK mTOR PIP5KαPIP5Kβ I 435 128 1 14 >10³ 978 6,729 >10³ >10³ >10³ NVP-BEZ-235 19 29363 267   3 6 1   2 ND* ND Novartis InvitroGen Adapta assay *ND = notdetermined

Example 16 No Off-Target Activity of Compound I in Kinome-Wide ProteinKinase Screen

This example demonstrates that compound I has little or no off targetactivity in a kinome-wide protein kinase screen. Using Ambit KINOMEscan™a genome wide screen of over 350 protein kinases failed to detect anyactivity at 10 μM. Examples of some kinases in the screen are shownbelow in Table 3.

TABLE 3 Examples of Relevant Kinases in Screen ABL FGFR1 JAK1 P38MAPKS6K AKT VEGFR1 JAK2 PDGFR SLK ALK FLT3 JNK1 PIM SRC BLK FRK KIT PKA SYKBRAF FYN LCK PKC TAK BTK HCK LYN PLK TIE CDK HER2 MAPK RAF TRK CSF1R ICKMEK RET TYK EGFR IGF1-R MET ROCK YES EPH ITK MLK ROS ZAP70

Example 17 Selectivity of Compound I for p110δ

This example demonstrates that compound I is selective for p110δ asmeasured in isoform specific cell-based assays.

Swiss-3T3 fibroblasts and RAW-264 were seeded on a 96-well tissueculture plate and allowed to reach at least 90% confluency. Cells werestarved and treated with either vehicle or serial dilutions of compoundI for 2 hrs and stimulated with PDGF or C5a respectively. Aktphosphorylation and total AKT was detected by ELISA. Purified B-cellswere treated with either vehicle or serial dilutions of compound I for30 minutes at room temperature before the addition of purified goatanti-human IgM. Results are expressed as relative [³H] thymidineincorporation induced by IgM crosslinking.

TABLE 4 PI3Kα PI3Kδ PI3Kγ EC₅₀ (nM) EC₅₀ (nM) EC₅₀ (nM) Fibroblast CellLine Primary B Cell Monocyte Cell Line PDGF induced pAKT BCR mediatedC5a induced pAKT proliferation >20,000 6 3,894 (n = 12) (n = 6) (n = 11)

Example 18 Expression of p110δ in Leukemia and Lymphoma Cell Lines

This example demonstrates that PI3K p110δ is highly expressed in a broadrange of leukemia and lymphoma cell lines.

PI3K p110δ promotes proliferation and survival in a wide range ofleukemia and lymphoma cell lines. Among the cell types investigated areMCL, DLBCL, AML, ALL, and CML.

Expression of PI3K p110 α, β, γ and δ in a panel of lymphoma andleukemia cell lines is demonstrated in FIG. 15. Proteins from 10⁶ cellswere separated by SDS-PAGE and analyzed by Western blot using antibodiesspecific for the α, β, γ and δ isoforms. Purified recombinant p110proteins were used as controls. Anti-actin antibodies were used toassess equal loading of the samples. p110δ was consistently expressed ata high level while other p110 isoforms were highly variable. PI3K p110δis known to be uniformly expressed in patient AML cells as discussed bySujobert, et al., Blood 2005 106(3), 1063-1066.

Example 19 Inhibitory Effect of Compound I on p110δ

Example 19 shows compound I inhibition of p110δ blocks PI3K signaling inleukemia and lymphoma cell lines with constitutive pathway activation.

The PI3K pathway is frequently deregulated in leukemia and lymphoma celllines. 48% of cell lines, or 13 out of 27, were found to haveconstitutive p-AKT. In addition, PI3K pathway activation is dependent onp110δ. Compound I was found to inhibit constitutive AKT phosphorylationin 13 out of 13 cell lines.

PAGE results of FIG. 9 demonstrates that constitutive AKTphosphorylation was inhibited by the presence of compound I in each of11 cell lines, including B-cell and T-cell lymphomas. Cells wereincubated for 2 hrs with 10 μM compound I. Cell lysates were run onSDS-PAGE and transferred onto PDVF membrane and probed with appropriateantibodies. Compound I was found to inhibit constitutive AKTphosphorylation in 11 out of 11 cell lines. Additional cell line datafor T-ALL and B-ALL cell lines is shown in FIG. 27. A decrease in Aktand S6 phosphorylation after exposure to a range concentrations ofcompound I (0.1 μM to 10 μM), was quantitated by densitometry, expressedas the percent change, FIG. 28A-B.

Example 20 Compound I Inhibits Proliferation and Apoptosis in LeukemiaCell Lines

Example 20 demonstrates that compound I inhibits proliferation andinduces apoptosis in leukemia cell lines. FIG. 16A-B show that treatmentwith compound I for 24 hours reduces cellular viability in a dosedependent manner.

Proliferation assays (AlamarBlue®) on ALL cell lines grown in thepresence of 10% FBS serum and measurements were taken at 24 hrs.Proliferation was measured in triplicate wells in 96-well plates. Theinhibition of PI3K signaling by compound I resulted in a block of cellcycle progression, and/or cell death. In each of six leukemia celllines, viability was reduced by 40-50% with 10 micromolar concentrationsof Compound I, FIG. 16A.

Induction of apoptosis by compound I. Cells were treated with DMSO(vehicle), 1 μM or 10 μM compound I for 24 hrs. The percentage ofapoptotic cells was determined by Annexin V-FITC/7AAD staining. Oneexperiment representative of different experiments that gave similarresults is shown in FIG. 16B.

Example 21 Expression of p110 Delta in CLL Cells

This example demonstrates PI3K p110δ and p110 δ isoform expression inpatient CLL cells.

PI3K mediated signaling pathways have been implicated in CLL. Thesepathways have a role in cell proliferation, prevention of apoptosis andcell migration. Efforts were made to determine PI3K isoform expressionin patient CLL cells.

CLL patient demographics are summarized below in Table 5.

TABLE 5 CLL Patient Demographics (Total (N = 24) I) Cytogeneticabnormalities 13q14.3 58% 11q22.3 33% 17p13.1 20% Trisomy 12 12% II)Treatment History Fludarabine refractory 29% Unknown 54% II) IgVH StatusMutated 33% Unmutated 33% Unknown 33%

The PAGE images of FIG. 17A-D compare the expression of p110α, p110δ,p110β, and p110γ in CLL cells of patients A-E. p110δ and p110γ isexpressed in each patient compared to the other PI3K isoforms.

Example 22 Compound I Induces Cleavage of Caspase 3 and PARP

This example demonstrates that compound I induced the cleavage ofcaspase 3 and PARP. FIG. 18A-B show results of caspase 3 and PARP(Poly(ADP) Ribose Polymerase) cleavage in the presence of 1, 10 μM ofcompound I or 25 μM of LY294002.

Further experiments provide evidence of compound I inducing caspase 2and PARP cleavage. Cells were cultured with compound I or vehicle alonefor 24 hrs. Thereafter, cells were lysed and sized-fractionated andimmunoblotted with antibody directed against FLIP, FIG. 29.Additionally, whole cell lysates were added to MDS (Meso ScaleDiagnostics) multi-spot 96-well 4 spot plates coated with Totalcaspase-3, cleaved caspase-3, cleaved PARP, and BSA. Proteins weredetected with antibodies labeled with SULFO-TAG reagent and quantified.A dose dependent response in the cleavage of caspase 3 and PARP wasachieved upon exposure to 5 or 10 μM of compound I.

Example 23 Compound I Blocks PI3K Signaling

This example demonstrates that compound I blocks PI3K signaling inpatient AML cells. PI3Kδ is implicated in signaling in AML patientcells. FIG. 21 shows the results of Phospho-Akt production in theabsence or presence of 0.1, 1.0, 10 μM of Compound I. This providesevidence that compound I reduces phopsho-Akt production in patient AMLcells.

Example 24 Measurement of PI3K Signaling in Basophils FoundingWhole-Blood

This example demonstrates a whole-blood assay for measurement of PI3Ksignaling in basophils using flow cytometry by the induction of CD63surface expression.

Inhibition of PI3K signaling in basophils permits compound I to be auseful pharmacodynamic marker. PI3K signaling is monitored by CD63surface expression. In particular, p110δ mediates FCεR1 signaling andp110γ mediates fMLP receptor signaling. The flow cytometry analysis ofPI3K mediated CD63 expression on basophils comprises the followingsequential steps:

1. Collect peripheral blood

2. Basophil stimulation (fMLP or Anti-FCεR1 Mab)

3. Label basophils (Anti-CCR3-FITC and Anti-CD63-PE)

4. Lyse and fix cells

5. Analysis by flow cytometry

FIG. 22A-C compares the results of A) no stimulation, B) stimulationwith Anti-FCεR1, or C) stimulation with fMLP.

FIG. 23 shows that Compound I is especially active where p110δ mediatedsignaling is most important, but is also relatively active where p110γis utilized: it achieved 50% reduction in SD63 expression at <<1 μM forthe p110δ test, and ca. 10 μM for the p110γ test. Basophil activationwas measured in human whole blood using the Flow2 CAST® kit. Whole bloodsamples were treated with either vehicle or serial dilutions of compoundI prior to activation of basophils either with anti-FcεRI mAb or fMLP.Cells were stained with the combination of anti-human CD63-FITC andanti-human CCR3-PE mAbs. The percent CD63 positive cells within thegated basophil population were determined in different treatment groupsand normalized to the vehicle control.

Example 25 Compound I Reduces Lymphadenopathy in CLL Patient Example 26

This example provides evidence of the reduction in size of a bulkylymphadenopathy in a CLL patient with a del[17p]. A patient withdel(17p) had an axillary lymphadenopathy, which was imaged by computedtomography (CT) to provide a baseline measurement of 5.9 cm×4.1 cm, FIG.40A. After one cycle of treatment with compound I, the lymphadenopathywas reduced to a dimension of 3.8×1.8 cm, FIG. 40B. A cycle treatmentwas 28 days of continuous oral dosing at either 200 mg BID or 350 mg BIDof compound I.

Limited Effect of Compound I on Glucose and Insulin Levels of a Subject

This example demonstrates that treatment with compound I has little orno effect on glucose and insulin levels. Compound I was administered at50-200 mg amounts BID to a subject over a period of up to 10 days. Bloodglucose and insulin concentrations were measured over time and comparedto placebo results as shown in FIG. 25A-B.

Blood glucose concentration remained steady after 10 days of treatmentwith even the highest dosage amount of compound I. Insulin levelsremained within the normal range after 7 days of treatment with compoundI. This provides evidence that compound I has little or no effect onglucose and insulin levels.

Example 27 Materials and Methods

This example provides information on materials and methods of carryingout the experiments described in Examples 28-35 which relate to the useof compound I in the treatment of multiple myeloma.

Materials

p110δ inhibitor compound I and compound II were provided by CalistogaPharmaceuticals, (Seattle, Wash.). The sample of compound I and II usedwas over 95% the S enantiomer. Compound I was dissolved in Dimethylsulphoxide at 10 mM and stored at −20° C. for in vitro study. CompoundII was dissolved in 1% carboxyl methylcellulose (CMC)/0.5% Tween 80 andstored at 4° C. for in vivo study. Recombinant human P110α, β, γ, and δwere reconstituted with sterile phosphate-buffered saline (PBS)containing 0.1% BSA. bortezomib was provided by MillenniumPharmaceuticals (Cambridge, Mass.). 3-Methyladenine was purchased fromSigma-Aldrich (St. Louis, Mo.).

Cell Culture

Dex-sensitive (MM.1S) and resistant (MM.1R) human MM cell lines werekindly provided by Dr. Steven Rosen (Northwestern University, Chicago,Ill.). H929, RPMI8226, and U266 human MM cell lines were obtained fromAmerican Type Culture Collection (Manassas, Va.). Melphalan-resistantRPMI-LR5 and Doxorubicin (Dox)-resistant RPMI-Dox40 cell lines werekindly provided by Dr. William Dalton (Lee Moffitt Cancer Center, Tampa,Fla.). OPM1 plasma cell leukemia cells were provided by Dr. EdwardThompson (University of Texas Medical Branch, Galveston). IL-6-dependenthuman MM cell line INA-6 was provided by Dr. Renate Burger (Universityof Kiel, Kiel, Germany) LB human MM cell line was established in thelaboratory. Phenotypic analysis revealed no cytogenetic abnormalities.Phenotypic analysis is shown in table 6. CD expression profile of LBcell line, defined by flow-cytometric analysis.

TABLE 6 LB expression CD marker % expression CD3 5.5% CD19 61.7% CD2097.2% CD38 54.1% CD40 96.8% CD49e 5.9% CD70 98.0% CD138 96.3%

All MM cell lines were cultured in RPMI1640 medium. Bone marrow stromalcells (BMSCs) were cultured in Dulbecco's modification of Eagle's medium(DMEM) (Sigma) containing 15% fetal bovine serum, 2 mM L-glutamine (LifeTechnologies), 100 U/mL penicillin, and 100 μg/mL streptomycin (LifeTechnologies). Blood samples collected from healthy volunteers wereprocessed by Ficoll-Paque™ gradient to obtain peripheral bloodmononuclear cells (PBMNCs). Patient MM and BM cells were obtained fromBM samples after informed consent was obtained per the Declaration ofHelsinki and approval by the Institutional Review Board of theDana-Farber Cancer Institute (Boston, Mass.). BM mononuclear cells wereseparated using Ficoll-Paque™ density sedimentation, and plasma cellswere purified (>95% CD138+) by positive selection with anti-CD138magnetic activated cell separation micro beads (Miltenyi Biotec, Auburn,Calif.). Tumor cells were also purified from the BM of MM patients usingthe RosetteSep negative selection system (StemCell Technologies,Vancouver, BC, Canada).

Growth Inhibition Assay

The growth inhibitory effect of compound I on growth of MM cell lines,PBMCs, and BMSCs was assessed by measuring3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetra-sodium bromide (MTT;Chemicon International, Temecula, Calif.) dye absorbance.

Effect of Compound I on Paracrine MM Cell Growth in the BM

MM cells (2×104 cells/well) were cultured for 48 h in BMSC coated96-well plates (Costar, Cambridge, Mass.), in the presence or absence ofdrug. DNA synthesis was measured by [3H]-thymidine (Perkin-Elmer,Boston, Mass.) uptake, with [3H]-thymidine (0.5μ, Ci/well) added duringthe last 8 h of 48 h cultures. All experiments were performed inquadruplicate.

Transient Knockdown of P110δ Expression

INA-6 cells and LB cells were transiently transfected with siRNAON-TARGET plus SMART pool P110δ or nonspecific control duplex (DharmaconLafayette, Co) using Cell Line Nucleofector Kit V (Amaxa BIosystemsGaitherburg, Md.).

Immunofluorescence

Viable MM cells (2.5×104) were pelleted on glass slides bycentrifugation at 500 rpm for 5 minutes using a cytospin system (ThermoShandon, Pittsburgh, Pa.). Cells were fixed in cold absolute acetone andmethanol for 10 min. Following fixation, cells were washed inphosphate-buffered saline (PBS) and then blocked for 60 min with 5% PBSin PBS. Slides were then incubated with anti-CD138 antibody (Santa CruzBiotechnology, Santa Cruz, Calif.) at 4° C. for 24 h, washed in PBS,incubated with goat anti-mouse IgG for 1 h at 4° C., and analyzed usingNikon E800 fluorescence microscopy.

Detection and Quantification of Acidic Vesicular Organelles (AVO) withAcridine Orange Staining.

Autophagy was characterized by sequestration of cytoplasmic proteins anddevelopment of AVOs. To detect and quantify AVOs in compound I or3MA-treated cells, vital staining was performed for 15 min with acridineorange at a final concentration of 1 μg/ml. Samples were examined undera fluorescence microscope.

Angiogenesis Assay

The anti-angiogenic activity of compound I was determined using an invitro Angiogenesis Assay Kit (Chemicon, Temecula, Calif.). HUVEC andendothelial growth media were obtained from Lonza (Walkersville, Md.,USA). HUVEC were cultured with compound I on polymerized matrix gel at37° C. After 8 h, tube formation was evaluated using Leika DM ILmicroscopy (Leica Microsystems, Wetzlar, Germany) and analyzed with IM50software (Leica Microsystems Imaging Solutions, Cambridge, UK). HUVECcell migration and rearrangement was visualized, and the number ofbranching points counted.

Western Blotting

MM cells were cultured with or without compound I; harvested; washed;and lysed using radioimmuno precipitation assay (RIPA) buffer, 2 mMNa₃VO₄, 5 m M NaF, 1 mM phenylmethylsulfonyl fluoride (5 mg/ml).Whole-cell lysates were subjected to sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation,transferred to Pure Nitrocellulose membranes (Bio-Rad Laboratories,Hercules, Calif.), and immunoblotted with anti-AKT, phospho(p)-AKT(Ser473, Thr 308), ERK1/2, P-ERK1/2, P-PDK1, STAT, P-STAT, P-FKRHL,P-70S6K, LC3, and PI3K/p110 α Abs (Cell Signaling Danvers, Mass.);anti-p110β, PI3K/p110δ, Glyceraldehyde 3-phosphate dehydrogenase(GAPDH), α-tubulin, and actin Abs (Santa Cruz Biotechnology, CA); andanti-p110 γ Ab (Alexis, San Diego, Calif.): and anti-LC3 Ab (Abgent, SanDiego, Calif.).

ELISA

Cytokine secretion by human BMSCs cocultured with MM cells was assessedby ELISA. BMSCs were cultured in 96-well plates with varyingconcentrations of compound I, with or without INA-6 cells. After 48 h,supernatants were harvested and stored at −80° C. Cytokines weremeasured using Duo set ELISA Development Kits (R&D Systems, Minneapolis,Minn.). All measurements were carried out in triplicate.

Human Cytokine Array

The cytokine levels in culture supernatants were assessed using ProteomeProfiler Antibody Arrays Panel A (R&D Systems, Minneapolis, Minn.),Supernatants from co-cultures with BMSCs were incubated for 4 hours withmembranes arrayed with Abs against 37 cytokines, according tomanufacturer's instructions.

Murine Xenograft Models of Human MM

CB17 SCID mice (48-54 days old) were purchased from Charles RiverLaboratories (Wilmington, Mass.). All animal studies were conductedaccording to protocols approved by the Animal Ethics Committee of theDana-Farber Cancer Institute. Mice were inoculated subcutaneously in theright flank with 3×106 LB cells in 100 μL RPMI-1640. When tumors werepalpable, mice were assigned into the treatment groups receiving 10mg/kg or 30 mg/kg gavages twice daily; and 7 mice in the control groupreceiving vehicle alone. Caliper measurements of the longestperpendicular tumor diameters were performed every alternate day toestimate the tumor volume using the following formula representing the3D volume of an ellipse: 4/3×(width/2)2×(length/2). Animals weresacrificed when tumors reached 2 cm or the mice appeared moribund.Survival was evaluated from the first day of treatment until death.Tumor growth was evaluated using caliper measurements from the first dayof treatment until day of first sacrifice, which was day 12 for thecontrol group and days 17 and 19 for the treatment groups. The imageswere captured with a canon IXY digital 700 camera. Ex vivo analysis oftumor images was captured with a LEICA DM IL microscope and LEICA DFC300FX camera at 40 u/0.60 (Leica, Heidelberg, Germany).

Human fetal bone grafts were implanted into CB17 SCID-mice (SCID-hu).Four weeks following bone implantation, 2.5×106 INA-6 cells wereinjected directly into the human BM cavity in the graft in a finalvolume of 100 μl of RPMI-1640 medium. An increase in the levels ofsoluble human IL-6 receptor (shuIL-6R) from INA-6 cells was used as anindicator of MM cell growth and burden of disease in SCID-hu mice. Micedeveloped measurable serum shuIL-6R approximately 4 weeks followingINA-6 cell injection, and then received either 10 or 30 mg/kg drug orvehicle alone daily for 7 weeks. Blood samples were collected andassessed for shuIL-6R levels using an enzyme-linked immunosorbent assay(ELISA, R&D Systems. Minneapolis Minn.).

Statistical Analysis

Statistical significance was determined by Dunn's multiple comparisontests. The minimal level of significance was p<0.05. Survival wasassessed using Kaplan-Meier curves and log-rank analysis. The combinedeffect of compound I and bortezomib was analyzed by isobologram analysisusing the CalcuSyn software program (Biosoft, Ferguson, Mo.); acombination index (CI)<0.7 indicates a synergistic effect.

Example 28 Expression of p110 Delta in MM Cells

This example demonstrates that p110 delta is highly expressed in patientMM cells. To assess PI3K/p110 expression, Abs was used againstrecombinant human PI3K/p110α, β, γ, and δ proteins with specificimmunoreactivity against these isoforms. The expression of p110δ in 11MM cell lines (MM.1S, OPM1, OPM2, RPMI8226, DOX40, LR5, MM.1R, U266,INA-6, H929, and LB), as well as 24 patient MM samples were evaluatedand immunoblots shown in FIG. 30A and FIG. 30B. FIG. 30A showsexpression of p110-α,-β, -γ, and -δ in MM cell lines detected byimmunoblotting using specific antibodies. Anti-α-Tubulin MAb served as aloading control. p110δ in patient MM cells was detected byimmunoblotting using anti-P110β Ab (FIG. 30B).

Anti-GAPDH MAb served as a loading control. INA-6 and LB cells stronglyexpressed p110δ, whereas MM.1S, OPM1, MM.1R, Dox40, U266 or H929 lackedp110δ expression (FIG. 30A).

p110δ expression in MM.1S and LB cells was confirmed byimmunofluorescence analysis (FIG. 30C). Human recombinantP110-α,-β,-γ,-δ proteins in SDS sample buffer were heated for 3 minprior to loading on gel. (10-20 μg per lane.) Recombinant humanP110-α,-β,-γ,-δ proteins were detected by Immunoblot analysis. Levels ofP110 δ were measured in MM1S and LB cells using P110 δ specific FITCconjugated secondary antibodies. P110δ stained green, and nucleic acids(DAPI) stained blue.

Western blotting revealed no correlation of between p110δ expression andexpression of the other isoforms (α, β and γ). Importantly, all patientMM cells also expressed p110δ, (FIG. 30B).

Example 29 Cytotoxicity of Compound I on MM Cells

This example demonstrates that compound I has selective cytotoxicityagainst cells with p110δ. Specifically, compound I potently inducedcytotoxicity in p110 delta positive MM cells as well as in primarypatient MM cells without cytotoxicity in peripheral blood mononuclearcells from healthy donors, suggesting a favorable therapeutic index.

The growth inhibitory effect of p110δ knockdown in MM cells wasevaluated. LB and INA-6 cells were transfected with P110δ siRNA (Si) orcontrol siRNA (Mock). After 24 h, expression of P110 δ was determined bywestern blot analysis, see FIG. 31A. INA-6 cells were transfected withp110δ siRNA or control siRNA, and then cultured for 72 hours. Cellgrowth was assessed by MTT assay, see FIG. 31 B. Data indicates mean±SDof triplicate cultures, expressed as fold of control. Transfection withp110δ siRNA, but not mock siRNA, down-regulated p110δ and inhibited MMcell growth at 72 h (FIG. 31A and FIG. 31B). The growth inhibitoryeffect of p110δ specific small molecule inhibitor compound I in MM celllines, PBMCs, and patient MM cells was evaluated.

Compound I induced cytotoxicity against LB and INA-6 MM cells(p110δ-positive) in a dose- and time-dependent fashion; in contrast,minimal cytotoxicity was noted in p110δ-negative cell lines (FIG. 31C).The legend for FIG. 31C: LB (□), INA-6 (Δ), RPMI 8226(∘), OPM2 (⋄), H929(●), U266 (♦), RPMI-LR5 (▴) and OPM1 (▪) MM cells were cultured with orwithout compound I for 48h.

Importantly, compound I also induced cytotoxicity against patient MMcells (FIG. 31D), without cytotoxicity in PBMCs from 4 healthyvolunteers at concentrations up to 20 μM (FIG. 31E). Patients MM cellsisolated from BM by negative selection were cultured with compound I for48h. Peripheral blood mononuclear cells isolated from healthy donorswere cultured with compound I for 72 h. Data represent mean±SDviability, assessed by MTT assay of triplicate cultures, expressed aspercentage of untreated controls. These results strongly suggest thatsensitivity to compound I is associated with P110δ expression, andsuggest a favorable therapeutic window.

To determine whether the cytotoxicity induced by compound I is viaapoptosis, the cleavage of caspases and PARP by western blot analysiswas examined. INA-6 cells were cultured with compound I (0-5 μM) for 120h. Total cell lysates were subjected to immunoblotting usinganti-caspase-3, -8, -9, PARP, and α-tubulin Abs. FL indicatesfull-length protein, and CL indicates cleaved protein. Significantlyincreased cleavage of caspase-8, caspase-9, caspase-3, and PARP wasobserved in INA-6 MM cells treated with compound I for 120 h (FIG. 31F).These results indicate that cytotoxicity triggered by compound I ismediated, at least in part, via caspase-dependent (both intrinsic andextrinsic) apoptosis.

Example 30 Inhibition of AKT and ERK Phosphorylation by Compound I

This example demonstrates the Inhibition of AKT and ERK phosphorylationby compound I.

An important downstream effector of PI3K is the serine/threonine proteinkinase AKT, which is activated by phosphorylation of Thr308 in theactivation loop of the kinase domain and Ser473 in the C-terminal tail.Phosphorylation of both sites requires an interaction between theN-terminal pleckstrin homology domain of AKT and membranephosphoinositide generated by PI3K. It was shown that compound Iinhibits both domains, suggesting that P110δ is the predominant isoformresponsible for PI3K signaling in MM cell lines.

Inhibition of AKT and ERK pathways in INA-6 cells by compound I wasexamined. INA-6 cells were cultured with Compound I or LY294002 for 12h, FIG. 32A. Actin Ab was used as a loading control. INA-6 and MM.1Scells were cultured with Compound I (0, 0.25, 1.0, 5.0 μM) for 6 hours,FIG. 32B. LB and INA-6 cells were cultured with compound I for 0-6hours, FIG. 32C. Whole cell lysates were subjected to immunoblottingusing AKT, P-AKT (Ser473 and Thr308), ERK1/2, P-ERK1/2, P-PDK1, andP-FKRHL antibodies. α-tubulin is used as a loading control.

Compound I significantly blocked phosphorylation of AKT and ERK1/2 inp110δ positive INA-6 cells (FIG. 32A), but did not affectphosphorylation of AKT or ERK in MM.1S cells with low expression ofP110δ (FIG. 32B). Compound I also significantly inhibitedphosphorylation of upstream PDK-1 and downstream FKHRL in INA-6 and LBMM cells in a time- and dose-dependent fashion (FIG. 32C), furtherconfirming inhibition of a both PI3K/AKT and ERK pathways in thesecells.

Example 31 Compound I Induces AVO Development and Autophagy

This example demonstrates the ability of compound I to trigger bothapoptosis and autophagy.

AKT regulates autophagy, thus investigation of compound I in inducingautophagy in LB and INA-6 MM cells was carried out.

INA-6 and LB MM cells were treated with 5 μM Compound I for 6h. CompoundI treatment induced LC3 accumulation in LB and INA-6 cells, evidenced byfluorescence microscopy or transmission electron microscopy.Autophagosome formation was defined by the accumulation of LC3; arrowsindicate autophagosomes, FIG. 33A.

INA-6 cells were treated with 5 μM Compound I or serum starvation for6h, stained with 1 μg/mL acridine orange for 15 min, and analyzed byfluorescence microscopy, FIG. 33B.

LC3 and beclin-1 protein levels were determined by western blottingusing LC3 and beclin-1 antibodies of lysates from INA-6 cells treatedwith Compound I, with or without 3-MA, FIG. 33C. GAPDH served as aloading control.

Immunofluorescence analysis showed markedly increased LC 3 staining inINA-6 and LB cells triggered by compound I (5 μM, 6 h) treatment (FIG.33A). Electron microscopic analysis also showed increased autophagicvacuoles (arrows) in MM cells treated with compound I. Since autophagyis characterized as acidic vesicular organelle (AVO) development,acridine orange staining was carried out. As shown in FIG. 33B, vitalstaining with acridine orange revealed development of AVOs in compoundI-treated LB and INA-6 cells. Moreover, markedly increased LC3-II andBeclin1 protein were detected in INA-6 MM cells after 6 h treatment withcompound I, which was blocked by 3-MA autophagic inhibitor (FIG. 33C).

No cytotoxicity in INA-6 and LB cells was induced by 3-MA atconcentrations up to 100 μM, FIG. 33D. P110 δ positive LB cells (♦) weretreated with 3-MA (0-100 μM) for 24h. Data represent means (±SD) oftriplicate cultures.

These results indicate that compound I induces development of AVOs andautophagy at earlier time points than induction of caspase/PARPcleavage.

Autophagy degrades cellular components, recycles cellular constituents,and responds to various cellular stress. In this example, LC3-II, ahallmark of autophagy, is induced by compound I treatment in p110 δpositive MM cell lines. Importantly, compound I treatment resulted in amarked increase in autophagy, evidenced by the presence of autophagicvacuoles in the cytoplasm, formation of AVOs, membrane association ofmicrotubule-associated protein I of LC3 with autophagosomes, and amarked induction of LC3-II protein. Electron microscopic analysisconfirmed that compound I induced autophagosomes. LC3-II was expressedthrough LC3-I conversion. Conversely, autophagy induced by compound Iwas suppressed by 3-MA, a specific inhibitor of autophagy. These studiessuggest that early cytotoxic effects of compound I are associated withautophagy.

Example 32 Compound I Inhibits Cell Growth in the Presence of BMSC

This example demonstrates the ability of compound I to inhibit paracrineMM cell growth with BMSCs.

Since IL-6 and IGF-1 induces growth and anti-apoptosis in MM cells,compound I was examined in overcoming the effects of these cytokines inINA-6 and LB MM cells. LB and INA-6 cells were cultured for 48h withcontrol media (▪); or with compound I at 5.0 μM (

) or 10 μM (□), in the presence or absence of IL-6 (1 and 10 ng/ml),FIG. 34A, or IGF-1 (10 and 100 ng/mL), FIG. 34B. DNA synthesis wasdetermined by measuring [3H]-thymidine incorporation during the last 8hof 72h cultures. Data represent means (±SD) of triplicate cultures.Neither IL-6 nor IGF-1 protected against the growth inhibition inducedby compound I (FIGS. 34A and B).

The BM microenvironment confers proliferation and drug-resistance in MM,thus MM cell growth inhibitory effect of compound I in the presence ofBMSCs was examined.

LB and INA-6 MM cells were cultured for 48h with control media (□), andwith 2.5 μM (

), 5 μM (

), and 10 μM (▪) of Compound I, in the presence or absence of BMSCs,FIG. 34C. DNA synthesis was determined by [3H]-thymidine incorporation.Data represent means (±SD) of triplicate cultures.

IL-6 in culture supernatants from BMSCs treated with compound I (0-2.5μM) was measured by ELISA, FIG. 34D. Error bars indicate SD (±).

BMSCs were cultured with 1.0 μM compound I or control media for 48h;cytokines in culture supernatants were detected using cytokine arrays,FIG. 34E.

INA-6 cells cultured with or without BMSCs were treated with compoundfor 48h. Total cell lysates were subjected to immunoblotting usingindicated antibodies, FIG. 34F. Actin was used as a loading control.

BMSCs from 2 different patients (□, ⋄) were cultured with compound I(0-20 μM) for 48h. Cell viability was assessed by MTT assay, FIG. 34G.Values represent mean±SD of triplicate cultures.

Importantly, compound I inhibited growth and cytokine secretion (FIG.34C-E), as well as phosphorylation of AKT and ERK (FIG. 34F), induced byBMSCs. In contrast, no significant growth inhibition in BMSCs was noted(FIG. 34G). These results indicate that compound I blocks paracrine MMcell growth in the context of the BM microenvironment.

Example 33 Compound I Inhibits Angiogenic HuVEC Tubule Formation

This example demonstrates the ability of Compound I to inhibit HuVECtubule formation. The role of PI3K, specifically p110 isoform, inangiogenesis was investigated. Endothelial cells are an essentialregulator of angiogenesis for tumor growth. Both Akt and ERK pathwaysare associated with endothelial cell growth and regulation ofangiogenesis; and importantly, endothelial cells express p110δ. Thisexample also demonstrates that compound I blocks in vitro capillary-liketube formation, associated with down regulation of Akt phosphorylation.

The effect of P110 δ inhibition on angiogenesis was investigated. HuVECswere treated with 0, 1.0, or 10 μM of compound I for 8 h, and tubeformation by endothelial cells was evaluated (FIG. 35A). HuVEC cellswere plated on Matrigel-coated surfaces and allowed to form tubules for8 h, in the presence or absence of Compound I. Endothelial cell tubeformation was measured by microscopic analysis, FIG. 35B. *P<0.005.

HuVECs were cultured with Compound I (0-20 μM) 48h, and viability wasassessed by MTT assay, FIG. 35C. Data shown are mean±SE of triplicatewells from a representative experiment. Thus, compound I inhibitedcapillary-like tube formation in a dose-dependent fashion (p<0.05) (FIG.35B), without associated cytotoxicity (FIG. 35C).

Phosphorylation and expression of AKT and ERK1/2 was markedly downregulated in HuVEC cells by compound I treatment. HuVECs were culturedwith compound I (0-200 μM) for 8h, and cell lysates were analyzed byimmunoblotting using the indicated antibodies, FIG. 35D. Actin was usedas a loading control.

These findings suggest that compound I can inhibit angiogenesis,associated with down regulation of AKT and ERK activity.

Example 34 Compound II Inhibits MM Cell Growth In Vivo

This example demonstrates the ability of compound II to inhibit human MMcell growth in vivo.

The in vivo efficacy of P110δ inhibitor was evaluated in a xenograftmodel in which SCID mice are injected subcutaneously with human MMcells.

Mice injected with 5×10⁶ LB cells were treated orally twice a day withcontrol vehicle (●), and compound II 10 mg/kg (□) or 30 mg/kg (∘). Meantumor volume was calculated as in Materials and Methods, FIG. 36A. Errorbars represent SD (±).

Representative whole-body images from a mouse treated for 12 d withcontrol vehicle (top panel) or Compound II (30 mg/kg) (bottom panel),FIG. 36B.

Tumors harvested from Compound II (30 mg/kg) treated mouse (right panel)and control mouse (left panel) were subjected to immuno-histochemistricanalysis using CD31 and P-AKT Abs. CD31 and P-AKT positive cells aredark brown, FIG. 36D.

Mice were treated with Compound II 10 mg/kg (- -), 30 mg/kg ( . . . ) orControl vehicle (-). Survival was evaluated from the first day oftreatment until sacrifice using Kaplan-Meier curves, FIG. 36C.

Tumor tissues were harvested from mice treated with control vehicle orCompound II (30 mg/kg). Protein levels of phosphorylated of PDK-1 andAKT (Ser473) were determined by western blotting of cell lysates, FIG.36E. Actin was used as a loading control.

Growth of INA-6 cells engrafted in human bone chips in SCID mice wasmonitored by serial serum measurements of shuIL-6R. Mice were treatedwith Compound II 10 mg/kg (□), 30 mg/kg (Δ) or control vehicle (●), andshuIL-6R levels were determined weekly by ELISA, FIG. 36F. Error barsindicate SD (±).

Compound II (p110 δ inhibitor) significantly reduced MM tumor growth inthe treatment group (n=7) compared with control mice (n=7). Comparisonof tumor volumes showed statistically significant differences betweencontrol versus treatment groups (vs 10 mg/kg, P<0.05; vs 30 mg/kg,P<0.01) (FIG. 36A). Marked decrease in tumor growth in treated versus incontrol mice was observed at day 12. (FIG. 36B) Kaplan-Meier curves andlog-rank analysis showed a mean Overall Survival (OS) of 15 days (95%confidence interval, 12-17 days) in control mice versus 23 days (95% CI,15-34 days) and 32 days (95% CI, 27-49 days) in the 10 mg/kg and 30mg/kg compound II treated groups, respectively. Statisticallysignificant prolongation in mean OS compared with control mice was alsoobserved in treatment groups (vs 10 mg/kg, P=0.086; vs 30 mg/kg,P=0.056) (FIG. 36C). Importantly, treatment with either the vehiclealone or compound II did not affect body weight. In addition,immunohistochemical (FIG. 36D) and immunoblot (FIG. 36E) analysisconfirmed that compound II treatment (30 mg/kg) significantly inhibitedp-Akt and p-PDK-1, as well as significantly decreased CD31 positivecells and microvessel density (p<0.01) (FIG. 36D). This suggests thatcompound II can inhibit angiogenesis in vivo via suppression of the Aktpathway.

In order to examine the activity of compound II on MM cell growth in thecontext of the human BM microenvironment in vivo, a SCID-hu model wasused in which IL-6 dependent INA-6 cells are directly injected into ahuman bone chip implanted subcutaneously in SCID-mice. This modelrecapitulates the human BM microenvironment with humanIL-6/BMSC-dependent growth of INA-6 human MM cells. These SCID-hu micewere treated with compound II or vehicle alone daily for 4 weeks, andserum shuIL-6R monitored as a marker tumor burden. As shown in FIG. 36F,compound II treatment significantly inhibited tumor growth compared withvehicle control. Significant tumor growth inhibition in this model wasobserved, evidenced by decreased serum shuIL-6R levels released by INA-6cells, confirming that p110δ inhibition blocks the MM growth promotingactivity of the BM microenvironment in vivo. Taken together, these datademonstrate that inhibition of p110δ by compound II significantlyinhibits MM growth in vivo and prolongs survival.

Example 35 Compound I in Combination with Bortezomib ExhibitsSynergistic Cytotoxicity

This example demonstrates the effect of Compound I in combination withbortezomib to mediate synergistic MM cytotoxicity.

The effects of combining compound I with bortezomib in inducingsynergistic MM cytotoxicity was investigated. LB and INA-6 MM cells werecultured with medium (▪) and with compound I, 1.25 μM (

), 2.5 μM (

), or 5.0 μM (□), in the presence or absence of bortezomib (0-5 nM).Cytotoxicity was assessed by MTT assay; data represent the mean±SD ofquadruplicate-cultures, FIG. 37A.

INA-6 cells were treated with Compound I (5 μM) and/or bortezomib (5 nM)for 6h. Phosphorylation of AKT was determined by western blotting ofcell lysates using phospho-AKT (ser473) antibody, FIG. 37B. Actin servedas a loading control.

Compound I enhances cytotoxicity of bortezomib. Increasingconcentrations of compound I (1.5-5.0 μM) added to bortezomib (2.5, 5.0nM) triggered synergistic cytotoxicity in LB and INA-6 MM cells (FIG.37A and Table 7). Importantly, induction of phospho-Akt by bortezomibtreatment was inhibited in the presence of compound I (FIG. 37B).

TABLE 7 Combination index (CI) Bortezomib Compound I (nM) (μM) Fa CI LB2.5 1.25 0.39 0.57 2.5 2.5 0.52 0.58 2.5 5 0.57 0.67 5 1.25 0.42 0.88 52.5 0.60 0.25 5 5 0.67 0.22 INA-6 2.5 1.25 0.49 0.31 2.5 2.5 0.58 0.482.5 5 0.69 0.54 5 1.25 0.56 0.73 5 2.5 0.66 0.42 5 5 0.75 0.31

Example 36 Compound I Effective in Follicular Lymphoma Cell Lines

This example provides evidence that compound I blocks PI3K signaling andinduces apoptosis in follicular lymphoma cells. P110δ is expressed in FLcell lines as shown in FIG. 38A. Certain cell lines show reduction inthe production of pAkt, Akt, pS6 and S6 when the cell is exposed tocompound I, FIG. 38B. Cleavage of PARP and Caspase-3 is observed afterexposure to compound I in a dose dependent fashion after 24 hours at 0.1μM and 0.5 μM, FIG. 38C.

Example 37 Compound I Effective in Primary MCL Cells

This example demonstrates that compound I is effective against MCL.Compound I was found to block constitutive PI3K signaling in primary MCLcells of two patients in a dose dependent manner when exposed to 0.1 μMor 1 μM of compound I, FIG. 39A. Compound I is also observed to inhibitsurvival factor and chemokine signaling in MCL cell lines. FIG. 39Bshows a significant reduction of pAkt in MCL lines exposed to differentsurvival factors in the presence of compound I.

Example 38 Effect of Compound in Combination with Ofatumumab in CLL

This example summarizes the Phase 1-2 study of repeated cycles (28days/cycle) of compound I in combination with ofatumumab for thetreatment of patients who had previously been treated for CLL.

Compound I (150 mg 2 times per day [BID]) was co-administeredcontinuously with 12 infusions of ofatumumab given over 24 weeks.Ofatumumab was administered with an initial dose of 300 mg on either Day1 or Day 2 (relative to the first dose of compound I). One week later,ofatumumab was administered at 1,000 mg every week for 7 doses, then at1,000 mg every 4 weeks for 4 doses. After completion of the ofatumumabtreatment, each subject continued to receive compound I as a singleagent at a dose of 150 mg BID as long as the subject was benefitting.

From the entire cohort of 21 patients, demographic and preliminaryefficacy data from 11 patients were available. The median [range] agewas 63 [54-76] years. The majority (9/11; 82%) of patients had bulkyadenopathy (≥lymph node measuring ≥5 cm in longest dimension). Themedian [range] number of prior therapies was 3 [1-6], including priorexposure to alkylating agents (10/11; 90%), rituximab (9/11; 82%),purine analogs (8/11; 72%), alemtuzumab (3/11; 28%) and/or ofatumumab(2/11; 18%). At the data cutoff, the median [range] treatment durationwas 5 [0-7] cycles. Almost all subjects (9/11; 82%) experienced markedand rapid reductions in lymphadenopathy within the first 2 cycles. Amongthe 11 patients, 10 were evaluable for response assessment at the end ofCycle 2 or later. Eight patients (80%) met criteria for a response asjudged by the investigator based on the criteria published in Hallek M,et al. (Guidelines for the diagnosis and treatment of chroniclymphocytic leukemia: a report from the International Workshop onChronic Lymphocytic Leukemia updating the National CancerInstitute-Working Group 1996 guidelines. Blood. 2008 Jun. 15;111(12):5446-56). One patient had reduced lymphadenopathy meetingcriteria for stable disease, and one patient had disease progression.The transient increase in the peripheral lymphocyte counts that wasexpected with single-agent PI3Kδ inhibition was reduced in magnitude andduration. The reduction of the transient lymphocytosis was induced bythe combination of compound I and oftatumumab.

Preliminary safety data show that the combination treatment had afavorable safety profile and lacked myelosuppression. In addition,pharmacodynamic data revealed that elevated baseline levels ofCLL-associated chemokines and cytokines (CCL3, CCL4, CXCL13, and TNFα)were reduced after 28 days of treatment. The results suggest that thecombination of compound I with ofatumumab provides a well-tolerated,non-cytotoxic treatment regimen in patients with previously treated CLL.

Example 39 Effect of Compound in Combination with BCL-2 Antagonists inCLL

This example shows the effect of compound I in combination with BCL-2antagonists ABT-737 and ABT-263 on the stroma-exposed CLL cells.

CLL Cell Purification:

Peripheral blood, bone marrow, and lymph node were obtained from consentpatients fulfilling diagnostic and immunophenotypic criteria for CLL.Peripheral blood mononuclear cells (PBMCs) were isolated from blood andtissue samples using the Ficoll-Paque (GE Healthcare, Waukesha, Wis.)density gradient centrifugation. Samples were either analyzed fresh orviably frozen in 10% dimethyl sulfoxide (DMSO; Sigma-Aldrich, St. Louis,Mo.) in fetal bovine serum (BD Biosciences, San Diego, Calif.) andstored in liquid nitrogen and later thawed for analysis. Single cellsuspensions were prepared for analysis on a fluorescence activated cellsorting (FACS) machine, and CD19+ CLL cells generally accounted for >85%of analyzed cells.

Cell Lines:

Murine CD154+ L cell line was maintained in RPMI 1640 mediumsupplemented with 10% FBS, 2.05 mM L-glutamine (HyClone, Logan, Utah),and penicillin-streptomycin (Cellgro, Manassas, Va.). The human stromalcell line StromaNKTert was purchased from the Riken cell bank (Tsukuba,Japan) and maintained in alpha-MEM supplemented with 1 μg/mLhydrocortisone, 10% PBS, 10% human serum (Invitrogen, Grand Island,N.Y.), 2.05-mM L-glutamine, and penicillin-streptomycin. Nurse-likecells (NLCs) were established by suspending PBMC from patients with CLLin complete RPMI 1640 medium with 10% FBS andpenicillin-streptomycin-glutamine to a concentration of 10⁷ cells/mL (2mL total). Cells were grown for 14 days in 24-well plates (Corning LifeSciences).

CLL Cell and Stromal Cell Co-Cultures:

CLL cells were cultured under standardized conditions on stromal celllines or primary NLC. Briefly, stromal cells were seeded one day priorto each experiment onto 24-well plates (Corning Life Sciences) at aconcentration of 3×10⁵ cells/mL/well and incubated at 37° C. in 5% CO₂.Stromal cell confluence was confirmed by phase contrast microscopy, andCLL cells were then added onto the stromal cell layer at a concentrationof 3×10⁶ cells/mL. Cultures were then treated with compounds for thespecified time periods. CLL cells were removed for analysis by gentlepipetting with media, and were then washed in PBS prior to analysis. A24-hour co-culture time point was used unless otherwise indicated.

Cell Viability Testing and Reagents:

CLL cell viability was determined by analysis of Annexin V-FITC (BDBiosciences, ^(San) Diego, Calif.) and Propidium Iodine (PI) (Sigma) byFACS. ABT-737, ABT-263, and compound I were stored in DMSO at −20° C.until use.

BH3 Profiling:

CLL patient peripheral blood, bone marrow, and lymph node samples wereanalyzed by either the plate-^(based) fluorimetry or FACS method.Briefly, PBMCs from CLL patients were made into single cell suspensionsand gently permeabilized using digitonin (0.002%). For thefluorimtery-based method, 100 μM JC-1 (Invitrogen) was added at thistime and cells were then loaded onto a 384-well plate, with individualwells containing individual BH3-only peptides. The JC1-BH3 assays werethen conducted in triplicate on a Tecan Safire 2 with Ex 545+/−20 nM andEm 590+/−20 nm with a three-hour time course. For the FACS-based method,single cell suspensions from CLL patient PBMCs were stained using humanFc Block (BD Pharmingen) followed by anti-CD19-V450 (BD Pharmingen) andanti-CXCR4-APC (BD Pharmingen). Cells were washed in PBS and then addedinto individual FACS tubes, each of which contained an individualBH3-only peptide. Samples were incubated at room temperature for 30minutes, 100 μM JC-1 was added to each tube, and the samples wereincubated for an additional 30 minutes. FACS measurements were conductedon a BD FACS Canto II with lasers at 407, 488, and 633 nm. JC-1 wasmeasured from the 488-nm laser using a 530/30-nm filter (FITC) and a585/42-nm filter (PE), and the degree of mitochondrial depolarizationwas calculated using the surrogate of the change in the median of PEsignal. The mitochondrial depolarization reported in response to eachBH3 peptide is normalized relative to the median percentage change in PEfluorescence of the JC-1 dye with a negative control, dimethyl sulfoxide(DMSO) (0%) and a positive control, the mitochondrial uncoupling agentcarbonyl cyanide 4-(trifluoromethoxy)-phenylhydrazone (FCCP) (100%).

Calcein-Based Adhesion Assay:

a confluent monolayer of CD154+L or StromaNKTert cells was generated byplating 1×10⁴ cells/well in 96-well plates for 24 hours. Single cellsuspensions of CLL cells at 0.5×10⁶ cells/ml were then labeled with 1μg/mL Calcein-AM (Invitrogen) for 1 hour. Cells ^(were) then spun downand treated with compound or vehicle for 1 to 24 hours. Non-adherentcells were washed off by aspiration. CLL cell adhesion was quantified byfluorimetry (Ex/Em=485/520 nm), and visualized directly using the NikonTE2000 inverted live-cell imaging system.

Data Analysis and Statistics:

Results are shown with standard error of mean and number of replicatesas described in each figure. Student's paired or unpaired t-tests,Mann-Whitney U test, or linear regression analyses were used forstatistical comparisons. Analyses were performed with GraphPad Prism 5software for PC (GraphPad Software, San Diego, Calif.). Flow cytometrydata were analyzed using FACS Diva version 6.1.1 (BD Pharmingen).Clinical response was assessed using 2008 IW-CLL criteria withresponders defined as patients achieving a complete or partial responseas best response, and non-responders as patients with stable disease,refractory disease, or progressive disease within 6 months of finishingfirst therapy. A two-tailed p-value≤0.05 was considered statisticallysignificant unless otherwise indicated.

Currently, many patients receiving the first-line traditional CLLtherapy often relapse and develop resistant to their treatment. BecauseCLL cells exposed to various stroma are resistant to treatment with bothcytotoxic chemotherapy (Kurtova A V et al. Diverse marrow stromal cellsprotect CLL cells from spontaneous and drug-induced apoptosis:development of a reliable and reproducible system to assess stromal celladhesion-mediated drug resistance. Blood. 2009; 114(20):4441-4450) andBH3-mimetics such as ABT-737 or ABT-263 (Vogler M et al. Concurrentup-regulation of BCL-XL and BCL2A1 induces approximately 1000-foldresistance to ABT-737 in chronic lymphocytic leukemia. Blood. 2009;113(18):4403-4413), CLL cells in the stromal microenvironment mayreceive proliferative or anti-apoptoic signals from stroma and becomeprotected from cell apoptosis. Thus, agents that antagonize theinteractions may reduce the stroma-mediated resistance in CLL.

The compound of formula I may modulate the stromal microenvironment. Inclinical studies, most patients treated with compound I and other agentstargeting the BCR pathway exhibited a rapid and transient lymphocytosis.Without being bound to any theory, compound I may modulate the stromalmicroenvironment by inhibiting CLL cell chemotaxis towards CXCL12/13,reducing CLL cell migration beneath stromal cells, down-regulatingchemokine secretion, or inhibiting phosphorylation of other downstreamtargets such as AKT and ERK. To characterize whether compound I modulatethe CLL-stroma interaction by increasing mitochondrial apoptosis orpriming, the BH3 profiling was used to measure the permeabilization ofmitochondria induced by peptides derived from the pro-death BH3 domainsof pro-death BCL-2 family proteins.

First, CLL cells from the peripheral blood of 30 patients were examinedMost patients had not been treated previously, and none had recentlyreceived therapy. At a final concentration of 0.03 μM in the BH3profiling, BIM BH3 peptide induced a significant amount ofdepolarization in most patient samples, with 22/30 (73.3%) of sampleshaving >50% depolarization by 1 hour. As shown in FIG. 41A, CLL cellswere highly primed for apoptosis. Also, the results of BH3 profilingshowed that most CLL patient samples showed relatively increaseddepolarization from BAD BH3 peptide, suggesting primary dependence onBCL-2 (n=23). As shown in FIG. 41B, some samples were observed to bemore dependent on MCL-1 (n=5), or BCL-XL (n=2). As shown in FIG. 41C,pre-treatment samples from treatment-naïve patients achieving a partialresponse (PR) or complete response (CR) by 2008 IW-CLL criteria wereobserved to be more primed than samples from patients with progressivedisease (PD) during or within 6 months of completing frontline CLLtherapy (p=0.024). As shown in FIG. 41D, BH3 profiling shows thatpatients with unmutated IGHV status (n=7) were significantly more primedthan patients with mutated IGHV status (n=18) (p=0.0026). As shown inFIG. 41E, percentage of VH homology to germline was observed to bepositively correlated with level of priming (p=0.0043). Thus, it wasobserved that CLL cells were highly primed for apoptosis, that CLL cellswere usually BCL-2 dependent, and that increased priming was associatedwith improved clinical response and unmutated IGHV.

Next, the effects of compound I on the adhesion, viability, and primingof the stroma-exposed CLL cells in vitro were evaluated. FIG. 42A-Egenerally show that compound I was observed to release CLL cellssequestered in stroma to overcome stroma-mediated resistance. Peripheralblood-derived CLL cells were labeled with calcein-AM and co-cultured onstromaNKTert for 24 hours with or without compound I (10 μM), rinsed bygentle pipetting, and visualized by wide-field microscopy. As shown inFIG. 42A, CLL cells co-cultured with stromaNKTert and treated withcompound I exhibited less adherent at 24 hours. Also, FIG. 42B showedthat the reduced adherence of CLL cells was detectable even after onlyone hour treatment of compound I, which was before CLL cell death wouldoccur. Moreover, FIG. 42C showed that the de-adherence of CLL cells fromstroma in response to compound I resulted in enhanced killing of CLLcells. In particular, mean percent viability for two patients wasdepicted along with SEM in FIG. 42C, and both of these patientsdemonstrated profound stroma-mediated resistance to either ABT-737 at100 nM or compound I at 10 μM alone. This resistance was observed to beovercome by the combination of the two compounds.

To avoid examine the direct killing of CLL cells by compound I, two CLLpatient samples that were resistant to both ABT-737 and compound I weretreated in the presence of stroma. In both samples, compound I restoredsensitivity of CLL cells to ABT-737 in the presence of stroma. Inaddition, stroma-exposed CLL cells treated with compound I (10 μM) incombination of various doses of ABT-737 and its oral analogue ABT-263showed that stroma-exposed CLL cells exhibited a dose-dependent increasein killing with either BH3 mimetic. In particular, with reference toFIG. 42D, resistance to ABT-737 was observed in the presence ofStromaNKTert, but may be overcome with concentrations of ABT-737 as lowas 10 nM. With reference to FIG. 42E, ABT-263 had a similardose-response curve.

To determine whether PI3K inhibition increased the sensitivity ofstroma-exposed CLL cells by increasing priming, PB-derived CLL cellscultured with or without StromaNKTert cells for 24 hours and wereexamined using Annexin-PI and BH3 profiling. Untreated CLL cellsgenerally exhibited apoptosis in ex vivo culture over 24 hours (CollinsR J et al. Spontaneous programmed death (apoptosis) of B-chroniclymphocytic leukaemia cells following their culture in vitro. Britishjournal of haematology. 1989; 71(3):343-350). Stromal co-culture of fourCLL patient samples led to protection from apoptosis in untreated cells.In particular, with reference to FIG. 43A, a two-way ANOVA analysisshowed that stroma provided protection from apoptosis in the absence ofcompound I. In the absence of stroma, compound I was observed to inducemore apoptosis than the control. In the presence of stroma, compound Iwas observed to induce significantly more apoptosis than the control.Thus, no significant difference was observed between killing by compoundI in the presence or absence of stroma.

However, the resistance or protection from apoptosis was reversed bycompound I. More than 40% of apoptosis were detected in stroma-exposedCLL cells treated with compound I (10 μM) compared to less than 10% ofapoptosis in untreated stroma-exposed CLL cells. Also, as shown in FIG.43B, the BH3 profiling showed that stroma-exposed CLL cells treated withcompound I exhibited an increased mitochondrial priming at 24 hourscompared to untreated cells (p=0.075). As shown in FIG. 43C, both BADBH3 peptide and ABT-737 used as a peptide induced significantly moremitochondrial depolarization in CLL cells treated with compound I(p=0.046 and p=0.047, respectively). This suggests that the treatmentwith compound I results in de-adherence of CLL from stroma, accompaniedby increased mitochondrial priming and increased sensitivity to BCL-2antagonism.

Overall, this example suggested that PI3K inhibition antagonized theprotection of CLL cells by stroma, and that compound I was effective atreversing the effects of stroma on CLL cells: adhesion, decreasedmitochondrial priming, and decreased sensitivity to therapies thatinhibit BCL-2. Also, the efficacy of compound I may be associated withlymphocyte redistribution in patients. By releasing CLL cells fromstroma, compound I likely allowed CLL cells to emerge from theanti-apoptotic stromal milieu, thereby increasing their mitochondrialpriming and being susceptible to apoptosis. This example also suggestedthe combinations of PI3K inhibition with BCL-2 inhibition increase theresponses to BCL-2 inhibition.

1. (canceled)
 2. A method for treating a solid tumor, comprisingadministering to a subject in need of such treatment an effective amountof a compound of formula A,

wherein R is H, halo, or C1-C6 alkyl; R′ is C1-C6 alkyl; or apharmaceutically acceptable salt thereof; and optionally apharmaceutically acceptable excipient.
 3. The method according to claim2, wherein the compound is predominantly the S-enantiomer.
 4. The methodaccording to claim 2, wherein R is fluoro (F) and is attached toposition 5 or 6 of the quinazolinyl ring.
 5. The method according toclaim 2, wherein R is H or F; and R′ is methyl, ethyl or propyl.
 6. Themethod according to claim 2, wherein the compound is


7. The method according to claim 2, wherein the compound is


8. The method according to claim 2, wherein the solid tumor is selectedfrom the group consisting of pancreatic cancer; bladder cancer;colorectal cancer; breast cancer, including metastatic breast cancer;prostate cancer, including androgen-dependent and androgen-independentprostate cancer; renal cancer, including metastatic renal cellcarcinoma; hepatocellular cancer; lung cancer, including non-small celllung cancer (NSCLC), bronchioloalveolar carcinoma (BAC), andadenocarcinoma of the lung; ovarian cancer, including progressiveepithelial or primary peritoneal cancer; cervical cancer; gastriccancer; esophageal cancer; head and neck cancer, including squamous cellcarcinoma of the head and neck; melanoma; neuroendocrine cancer,including metastatic neuroendocrine tumors; brain tumors, includingglioma, anaplastic oligodendroglioma, adult glioblastoma multiforme, andadult anaplastic astrocytoma; bone cancer; and soft tissue sarcoma. 9.The method according to claim 2, wherein the solid tumor is breastcancer.
 10. The method according to claim 9, wherein the breast canceris metastatic breast cancer.
 11. The method according to claim 2,wherein the solid tumor is ovarian cancer.
 12. The method according toclaim 6, wherein the solid tumor is selected from the group consistingof pancreatic cancer; bladder cancer; colorectal cancer; breast cancer,including metastatic breast cancer; prostate cancer, includingandrogen-dependent and androgen-independent prostate cancer; renalcancer, including metastatic renal cell carcinoma; hepatocellularcancer; lung cancer, including non-small cell lung cancer (NSCLC),bronchioloalveolar carcinoma (BAC), and adenocarcinoma of the lung;ovarian cancer, including progressive epithelial or primary peritonealcancer; cervical cancer; gastric cancer; esophageal cancer; head andneck cancer, including squamous cell carcinoma of the head and neck;melanoma; neuroendocrine cancer, including metastatic neuroendocrinetumors; brain tumors, including glioma, anaplastic oligodendroglioma,adult glioblastoma multiforme, and adult anaplastic astrocytoma; bonecancer; and soft tissue sarcoma.
 13. The method according to claim 6,wherein the solid tumor is breast cancer.
 14. The method according toclaim 13, wherein the breast cancer is metastatic breast cancer.
 15. Themethod according to claim 6, wherein the solid tumor is ovarian cancer.16. The method according to claim 2, further comprising administeringsimultaneously or separately (i) a therapeutically effective amount ofat least one additional therapeutic agent and/or (ii) a therapeuticprocedure.
 17. The method according to claim 16, wherein the at leastone additional therapeutic agent is a proteasome inhibitor.
 18. Themethod according to claim 17, wherein the proteasome inhibitor isselected from the group consisting of bortezomib, carfilzomib, PR-047,disulfiram, lactacystin, PS-519, eponemycin, epoxomycin, acalacinomycin,CEP-1612, MG-132, CVT-63417, PS-341, a vinyl sulfone tripeptideinhibitor, ritonavir, PI-083, (+/−)-7-methylomuralide, and(−)-7-methylomuralide.
 19. The method according to claim 16, wherein thetherapeutic procedure is selected from the group consisting of antibodytherapy, biological therapy, enzyme inhibitor therapy, total bodyirradiation, low-LET cobalt-60 gamma ray therapy, bleomycin,conventional surgery, radiation therapy, and high-dose chemotherapy. 20.The method according to claim 2, wherein the subject is refractory tochemotherapy treatment or is in relapse after treatment withchemotherapy.
 21. The method according to claim 2, wherein the compoundof formula A is present in a pharmaceutical composition comprising thecompound of formula A and at least one pharmaceutically acceptableexcipient.